Method of screening compounds for treating bladder cancer

ABSTRACT

Objective methods for detecting and diagnosing bladder cancer (BLC) are described herein. In one embodiment, the diagnostic method involves determining the expression level of a BLC-associated gene that discriminates between BLC cells and normal cells. The present invention further provides means for predicting and preventing bladder cancer metastasis using BLC-associated genes having unique altered expression patterns in bladder cancer cells with lymph-node metastasis. The present invention provides methods of screening for therapeutic agents useful in the treatment of bladder cancer, methods of treating bladder cancer and method for vaccinating a subject against bladder cancer. Specifically, the present application provides novel human genes C2093, B5860Ns and C6055s whose expression is markedly elevated in bladder cancers. The genes and polypeptides encoded by the genes can be used, for example, in the diagnosis of bladder cancers, as target molecules for developing drugs against the disease, and for attenuating cell growth of bladder cancer.

This application is a divisional of U.S. patent application Ser. No. 13/168,720, filed Jun. 24, 2011, which is a divisional of U.S. patent application Ser. No. 11/815,850, filed Nov. 20, 2008, now U.S. Pat. No. 7,998,695, which is a U.S. National Stage of PCT/JP2006/302684, filed Feb. 9, 2009, which claims the benefit of U.S. Provisional Application Ser. No. 60/652,318 filed Feb. 10, 2005 and U.S. Provisional Application Ser. No. 60/703,225 filed Jul. 27, 2005, the contents of which are hereby incorporated by reference in their entirety.

This application includes a Sequence Listing as a text file named “87331-012320US-844785_SEQLIST.txt” created Jun. 27, 2012, and containing 9,279,699 bytes. The material contained in this text file is incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to methods of detecting and diagnosing bladder cancer as well as methods of treating and preventing bladder cancer and bladder cancer metastasis. The present invention also relates to genes and polypeptides associated with bladder cancers.

BACKGROUND OF THE INVENTION

Bladder cancer is the second most common genitourinary tumor in human populations, with an incidence of approximately 261,000 new cases each year worldwide; about a third of those are likely to be invasive or metastatic disease at the time of diagnosis (Parkin D M, et al., (1999) CA Cancer J Clin; 49:33-64). Although radical cystectomy is considered the “gold standard” for treatment of patients with localized but muscle-invasive bladder cancer, about 50% of such patients develop metastases within two years after cystectomy and subsequently die of the disease (Sternberg C N., (1995) Ann Oncol; 6:113-26).

Neoadjuvant chemotherapy is usually prescribed for muscle-invasive bladder cancer to treat micrometastases and to improve resectability of larger neoplasms (Fagg S L, et al., (1984) Br J Urol; 56:296-300, Raghavan D, et al., (1984) Med J Aust; 140:276-8). Regimens involving methotrexate, vinblastine, doxorubicin, and cisplatin (M-VAC), followed by radical cystectomy, are more likely to eliminate residual cancer than radical cystectomy alone, and, as such, improve survival among patients with locally advanced bladder cancer ((2003) Lancet; 361:1927-34, Grossman H B, et al., (2003) N Engl J Med; 349:859-66). In some clinical trials, down-staging with drugs prior to surgery was shown to have significant survival benefits (Grossman H B, et al., (2003) N Engl J Med; 349:859-66, Splinter T A, et al., (1992) J Urol; 147:606-8); moreover, patients who respond to neoadjuvant chemotherapy may preserve bladder function and enjoy an improved quality of life. However, since no method yet exists for predicting the response of an individual patient to chemotherapies, such as M-VAC, some patients will suffer from adverse reactions to the drugs without achieving any benefit in terms of positive effects, often losing the opportunity for additional therapy when their physical condition deteriorates. Hence, it is of critical importance to identify molecular targets for the development of novel drugs for bladder cancer patients. Some recent studies have demonstrated that gene expression information generated by cDNA microarray analysis in human tumors can provide molecular phenotyping that identifies distinct tumor classifications not evident by traditional histopathological method (Armstrong, S. A, et al., (2002) Nat Genet, 30: 41-47; Golub, T. R, et al., (1999) Science, 286: 531-537; Hofmann, W. K et al., (2002) Lancet, 359: 481-486). Moreover, several studies have demonstrated the effectiveness of this method for identifying novel cancer-related genes. The promise of such information lies in the potential to improve clinical strategies with neoplastic disease.

SUMMARY OF THE INVENTION

Hence, in the study reported here, we identified novel molecular targets using genome-wide information obtained from 33 invasive bladder cancer cases on a cDNA microarray consisting of 27,648 transcribed elements in combination with laser microbeam microdissection (LMM) of the tumors to obtain pure populations of cancer cells for analysis. These results suggest that such information may lead ultimately to our goal of “personalized therapy”.

To characterize the detailed molecular mechanisms associated with bladder cancers, with a view toward development of novel therapeutic targets, the present inventors analyzed gene-expression profiles of 33 cancer cells using a cDNA microarray representing 27,648 genes coupled with laser microbeam microdissection (LMM). By comparing expression patterns between cancer cells from diagnostic bladder cancer patients and normal human bladder cells (used as universal control), 394 genes that were commonly up-regulated in bladder cancer cells were identified. Of those genes, 288 represent functionally characterized genes that were up-regulated in bladder cancer cells; however, the functions of the remaining 106 (including 51 ESTs) genes are currently unknown. In addition, 1272 genes were identified as being commonly down-regulated in bladder cancer cells. Of these, 1026 represent functionally characterized genes that were down-regulated in bladder cancer cells; however, the functions of the remaining 246 (including 119 ESTs) are currently unknown. The genes contained in the semi-quantitative RT-PCR experiments of representative 44 up-regulated genes supported the results of our microarray analysis. Accordingly, the data herein will provide useful information for finding candidate genes whose products may serve as molecular targets for treatment of bladder cancers.

The present invention is based on the discovery of a pattern of gene expression that correlates with bladder cancer (BLC). Genes that are differentially expressed in bladder cancer are collectively referred to herein as “BLC nucleic acids” or “BLC polynucleotides” and the corresponding encoded polypeptides are referred to as “BLC polypeptides” or “BLC proteins.”

Through the expression profiles of bladder cancers, the present inventors identified two specific genes, labeled C2093, B5860N and C6055, respectively, that were significantly overexpressed in bladder cancer cells. Furthermore, the present inventors isolated a novel transcriptional variant of the B5860N and C6055 gene. It was further demonstrated that the treatment of bladder cancer cells with siRNA effectively inhibited expression of C2093, B5860N and C6055 and suppressed cell/tumor growth of bladder cancer. These findings suggest that C2093, B5860N and C6055 play key roles in tumor cell growth, and, therefore, represent promising targets for the development of anti-cancer drugs.

The full-length mRNA sequence of C2093 contained 6319 nucleotides (SEQ ID NO: 1), encoding a polypeptide of 1780 amino acids (SEQ ID NO: 2). The B5860N gene has two different transcriptional variants, consisting of 12 and 11 exons and corresponding to B5860N V1 (SEQ ID NO. 3, encoding SEQ ID NO. 4) and B5860N V2 (SEQ ID NO. 5, encoding SEQ ID NO. 6), respectively (FIG. 3 b). There were alternative variations in exon 8 of V1; however, the remaining exons were common to both variants. The V2 variant does not have exon 8 of the V1, but does generate the same stop codon within last exon. The full-length cDNA sequences of the B5860NV1 and B5860NV2 variants consist of 5318 and 4466 nucleotides, respectively. The ORF of these variants start within each exon 1. The V1 and V2 transcripts ultimately encode polypeptides of 812 and 528 amino acids, respectively. Accordingly, the term “B5860Ns” as used herein, refers to either or both of transcripts of B5860NV1 and B5860NV2. Namely, in the context of the present invention, it was revealed that the B5860N gene may be expressed as at least two transcript variants. To further confirm the expression pattern of each variant in bladder cancer cell lines and normal human tissues, including bladder, heart, lung, liver, kidney, brain, and pancreas, the present inventors performed northern blot analysis. As a result, it was discovered that both variants were highly overexpressed in bladder cancer cells; however, expression in normal human tissues was either absent or undetectable (FIG. 2 f, lower panel). In particular, the V2 transcript was expressed exclusively in testis. The C6055 gene has four different splicing variants consisting of 24, 25, 22 and 22 exons, corresponding to MGC34032 (GeneBank Accession No. NM_(—)152697, SEQ ID NO: 133 encoding a polypeptide of SEQ ID NO: 134), Genbank Accession No. AK128063 (SEQ ID NO: 135 encoding a polypeptide of SEQ ID NO: 136, C6055V1 (SEQ ID NO:129 encoding a polypeptide of SEQ ID NO:130) and C6055V2 (SEQ ID NO:131 encoding a polypeptide of SEQ ID NO:132), respectively (FIG. 3 c). There were alternative variations in exon 1, 2, 3 and 24 of MGC34032, and the other remaining exons were common among four transcripts. C6055V1 and C6055V2 transcripts have no exon 1, 2 and 3 of MGC34032, generating same stop codon within last exon. Moreover, C6055V1, C6055V2 and Genbank Accession No. AK128063 transcripts have a different exon 24 of MGC34032. Genbank Accession No. AK128063 has a new exon as an exon 4a. In particular, the ORF of C6055V1 and C6055V2 transcripts start at within each exon 4, indicating C6055V1 and C6055V2 transcripts have same ORF. The full-length cDNA sequences of MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2 transcripts consist of 2302, 3947, 3851, and 3819 nucleotides, respectively. Eventually, MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2 transcripts encode 719, 587, 675 and 675 amino acids, respectively. Accordingly, the term “C6055s” as used herein, refers to one or more of transcripts of MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2. Namely, in the context of the present invention, it was revealed that the C6055 gene may be expressed as at least four transcript variants. To further confirm the expression pattern of each variant in bladder cancer cell lines and normal human tissues including bladder, heart, lung, liver, kidney, brain, testis, pancreas, we performed northern blot analysis. As a result, approximately 3.9 kb transcripts were highly overexpressed in some bladder cancer cells (HT-1376, SW780 and RT4), but no or undetectable expression in normal human tissues (FIG. 2 g). In addition, 7.5 kb transcript was specifically expressed only in HT1376 cells, but we have not yet identified the entire mRNA sequence of this transcript. Furthermore, when we performed northern blot analysis using the common region among these transcripts as a probe, we detected 2.3 kb transcript exclusively in normal testis, corresponding to MGC34032 (FIG. 2 h). Therefore, we further perform functional analysis for C6055V1 gene product.

Many anticancer drugs are not only toxic to cancer cells but also to normally growing cells. However, since the normal expression of C2093, B5860Ns and C6055s is restricted to the testis, agents that suppress the expression of C2093, B5860Ns and C6055s may not adversely affect other organs, and thus may be conveniently used for treating or preventing bladder cancer.

Thus, the present invention provides a novel transcriptional variant, B5860NV1, which serves as a candidate for a diagnostic marker for bladder cancer as well as a promising potential target for developing new strategies for bladder cancer diagnosis and effective anti-cancer agents. Furthermore, the present invention provides a polypeptide encoded by this gene, as well as methods for the production and use of the same. More specifically, the present invention provides a novel human polypeptide, B5860NV1, or a functional equivalent thereof, the expression of which is elevated in bladder cancer cells.

In a preferred embodiment, the B5860NV1 polypeptide includes an 811 amino acid (SEQ ID NO: 4) protein encoded by the open reading frame of SEQ ID NO: 3. The present application also provides an isolated protein encoded from at least a portion of the B5860NV1 polynucleotide sequence, or polynucleotide sequences that are at least 15%, more preferably at least 25%, complementary to the sequence set forth in SEQ ID NO: 3, to the extent that they encode a B5860NV1 protein or a functional equivalent thereof. Examples of such polynucleotides are degenerates and allelic mutants of B5860NV1 encoded by the sequence of SEQ ID NO: 3.

As used herein, an isolated gene is a polynucleotide the structure of which is not identical to that of any naturally occurring polynucleotide or to that of any fragment of a naturally occurring genomic polynucleotide spanning more than three separate genes. The term therefore includes, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule in the genome of the organism in which it naturally occurs; (b) a polynucleotide incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule, such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion polypeptide.

Accordingly, in one aspect, the invention provides an isolated polynucleotide that encodes a polypeptide described herein or a fragment thereof. Preferably, the isolated polynucleotide includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO: 3. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identical to the nucleotide sequence shown in SEQ ID NO: 3. In the case of an isolated polynucleotide which is longer than or equivalent in length to the reference sequence, e.g., SEQ ID NO: 3, the comparison is made with the full length of the reference sequence. Where the isolated polynucleotide is shorter than the reference sequence, e.g., shorter than SEQ ID NO: 3, the comparison is made to a segment of the reference sequence of the same length (excluding any loop required by the homology calculation).

The present invention also provides a method of producing a protein by transfecting or transforming a host cell with a polynucleotide sequence encoding the B5860NV1 protein, and expressing the polynucleotide sequence. In addition, the present invention provides vectors comprising a nucleotide sequence encoding the B5860NV1 protein, and host cells harboring a polynucleotide encoding the B5860NV1 protein. Such vectors and host cells may be used for producing the B5860NV1 protein.

A binding agent that specifically recognizes the B5860NV1 protein is also provided by the present application. For example, a binding agent may be an antibody raised against a B5860NV1 protein. Alternatively, a binding agent may be a ligand specific for the protein, or a synthetic polypeptide that specifically binds the protein (see e.g., WO2004/044011). An antisense polynucleotide (e.g., antisense DNA), ribozyme, and siRNA (small interfering RNA) of the B5860NV1 gene are also provided.

Accordingly, the present invention provides a method of diagnosing or determining a predisposition to bladder cancer in a subject by determining an expression level of a BLC-associated gene in a patient-derived biological sample, such as tissue sample. The term “BLC-associated gene” refers to a gene that is characterized by an expression level which differs in a BLC cell as compared to a normal cell. A normal cell is one obtained from bladder tissue. In the context of the present invention, a BLC-associated gene is a gene listed in Tables 4-5 (i.e., genes of BLC Nos. 1-1666). An alteration, e.g., an increase or decrease in the level of expression of a gene as compared to a normal control level of the gene, indicates that the subject suffers from or is at risk of developing BLC.

In the context of the present invention, the phrase “control level” refers to a protein expression level detected in a control sample and includes both a normal control level and a bladder cancer control level. A control level can be a single expression pattern derived from a single reference population or a value derived from a plurality of expression patterns. For example, the control level can be obtained from a database of expression patterns from previously tested cells. A “normal control level” refers to a level of gene expression detected in a normal, healthy individual or in a population of individuals known not to be suffering from bladder cancer. A normal individual is one with no clinical symptoms of bladder cancer. On the other hand, a “BLC control level” refers to an expression profile of BLC-associated genes found in a population suffering from BLC.

An increase in the expression level of one or more BLC-associated genes listed in Table 4 (i.e., the over-expressed or up-regulated genes of BLC Nos. 1-394) detected in a test sample as compared to a normal control level indicates that the subject (from which the sample was obtained) suffers from or is at risk of developing BLC. In contrast, a decrease in the expression level of one or more BLC-associated genes listed in Table 5 (i.e., the under-expressed or down-regulated genes of BLC Nos. 395-1666) detected in a test sample compared to a normal control level indicates said subject suffers from or is at risk of developing BLC.

Alternatively, expression of a panel of BLC-associated genes in a sample can be compared to a BLC control level of the same panel of genes. A similarity between sample expression and BLC control expression indicates that the subject (from which the sample was obtained) suffers from or is at risk of developing BLC.

According to the present invention, a gene expression level is deemed “altered” when expression of the gene is increased or decreased by at least 10%, preferably at least 25%, more preferably 50% or more as compared to the control level. Alternatively, an expression level is deemed “increased” or “decreased” when gene expression is increased or decreased by at least 0.1, at least 0.2, at least 1, at least 2, at least 5, or at least 10 or more fold as compared to a control level. Expression is determined by detecting hybridization, e.g., on an array, of a BLC-associated gene probe to a gene transcript of the patient-derived tissue sample.

In the context of the present invention, the patient-derived tissue sample may be any tissue obtained from a test subject, e.g., a patient known to or suspected of having BLC. For example, the tissue may contain an epithelial cell. More particularly, the tissue may be an epithelial cell from a bladder ductal carcinoma.

The present invention further provides a method for the diagnosis of bladder cancer which includes the step of determining an expression level of a C2093, B5860Ns or C6055s gene in a biological sample from a subject, comparing the expression level of the gene with that in a normal sample, and defining that a high expression level of the C2093, B5860Ns or C6055s gene in the sample indicates that the subject suffers from or is at risk of developing bladder cancer.

The present invention also provides a BLC reference expression profile, comprising a gene expression level of two or more of BLC-associated genes listed in Tables 4-5. Alternatively, the BLC reference expression profile may comprise the levels of expression of two or more of the BLC-associated genes listed in Table 4, or the BLC-associated genes listed in Table 5.

The present invention further provides methods of identifying an agent that inhibits or enhances the expression or activity of a BLC-associated gene, e.g. a BLC-associated gene listed in Tables 4-5, by contacting a test cell expressing a BLC-associated gene with a test compound and determining the expression level of the BLC-associated gene or the activity of its gene product. The test cell may be an epithelial cell, such as an epithelial cell obtained from a bladder carcinoma. A decrease in the expression level of an up-regulated BLC-associated gene or the activity of its gene product as compared to a normal control level or activity of the gene or gene product indicates that the test agent is an inhibitor of the BLC-associated gene and may be used to reduce a symptom of BLC, e.g. the expression of one or more BLC-associated genes listed in Table 4. Alternatively, an increase in the expression level of a down-regulated BLC-associated gene or the activity of its gene product as compared to a normal control level or activity of the gene or gene product indicates that the test agent is an enhancer of expression or function of the BLC-associated gene and may be used to reduce a symptom of BLC, e.g., the under-expression of one or more BLC-associated genes listed in Table 5.

Further, a method of screening for a compound for treating or preventing bladder cancer is provided by the present invention. The method includes contacting a C2093, B5860Ns or C6055s polypeptide with test compounds, and selecting test compounds that bind to or that alter the biological activity of the C2093, B5860Ns or C6055s polypeptide.

The present invention further provides a method of screening for a compound for treating or preventing bladder cancer, wherein the method includes contacting a test compound with a cell expressing a C2093, B5860Ns or C6055s polypeptide or introduced with a vector comprising a transcriptional regulatory region of C2093, B5860Ns or C6055s upstream of a reporter gene, and selecting the test compound that suppresses the expression level or activity of the C2093, B5860Ns or C6055s polypeptide or a reporter gene product.

The present invention also provides a kit comprising a detection reagent which binds to one or more BLC nucleic acids or BLC polypeptides. Also provided is an array of nucleic acids that binds to one or more BLC nucleic acids.

Therapeutic methods of the present invention include a method of treating or preventing BLC in a subject, including the step of administering to the subject an antisense composition. In the context of the present invention, the antisense composition reduces the expression of the specific target gene. For example, the antisense composition may contain a nucleotide which is complementary to a BLC-associated gene sequence selected from the group consisting of the up-regulated BLC-associated genes listed in Table 4. Alternatively, the present method may include the steps of administering to a subject a small interfering RNA (siRNA) composition. In the context of the present invention, the siRNA composition reduces the expression of a BLC nucleic acid selected from the group consisting of the BLC-associated genes listed in Table 4. In yet another method, the treatment or prevention of BLC in a subject may be carried out by administering to a subject a ribozyme composition. In the context of the present invention, the nucleic acid-specific ribozyme composition reduces the expression of a BLC nucleic acid selected from the group consisting of the BLC-associated genes listed in Table 4. Thus, in the present invention, the BLC-associated genes listed in Table 4 are preferred therapeutic targets for bladder cancer. Other therapeutic methods include those in which a subject is administered a compound that increases the expression of one or more of the down-regulated BLC-associated genes listed in Table 5 or the activity of a polypeptide encoded by one or more of the BLC-associated genes listed in Table 5.

The present invention further provides methods for treating or preventing bladder cancer using the pharmaceutical composition provided by the present invention.

In addition, the present invention provides methods for treating or preventing cancer, which comprise the step of administering a C2093, B5860Ns or C6055s polypeptide. It is expected that anti-tumor immunity will be induced by the administration of a C2093, B5860Ns or C6055s polypeptide. Thus, the present invention also provides a method for inducing anti-tumor immunity, which method comprises the step of administering a C2093, B5860Ns or C6055s polypeptide, as well as pharmaceutical compositions for treating or preventing cancer comprising a C2093, B5860Ns or C6055s polypeptide.

The present invention also includes vaccines and vaccination methods. For example, a method of treating or preventing BLC in a subject may involve administering to the subject a vaccine containing a polypeptide encoded by a nucleic acid selected from the group consisting of the BLC-associated genes listed in Table 4 or an immunologically active fragment of such a polypeptide. In the context of the present invention, an immunologically active fragment is a polypeptide that is shorter in length than the full-length naturally-occurring protein, yet which induces an immune response analogous to that induced by the full-length protein. For example, an immunologically active fragment should be at least 8 residues in length and capable of stimulating an immune cell, such as a T cell or a B cell. Immune cell stimulation can be measured by detecting cell proliferation, elaboration of cytokines (e.g., IL-2), or production of an antibody.

The present application also provides a pharmaceutical composition for treating or preventing bladder cancer. The pharmaceutical composition may be, for example, an anti-cancer agent. The pharmaceutical composition can comprise at least a portion of antisense S-oligonucleotides, siRNA molecules or ribozymes against the C2093, B5860Ns or C6055s polynucleotide sequences shown and described in SEQ ID NOs: 1, 3, 5, 129, 131, 133 and 135 respectively. A suitable siRNA targets a sequence of SEQ ID NO: 21, 25 or 144. Thus, an siRNA of the invention comprises a nucleotide sequence selected from SEQ ID NO: 21, 25 or 144. This may be preferably selected as targets for treating or preventing bladder cancer according to the present invention. The pharmaceutical compositions may be also those comprising the compounds selected by the present methods of screening for compounds for treating or preventing cell proliferative diseases, such as bladder cancer.

The course of action of the pharmaceutical composition is desirably to inhibit growth of the cancerous cells, such as bladder cancer cells. The pharmaceutical composition may be applied to mammals, including humans and domesticated mammals. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

One advantage of the methods described herein is that the disease is identified prior to detection of overt clinical symptoms of bladder cancer. Other features and advantages of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples, as well as the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a photograph of a DNA agarose gel showing expression of representative 44 genes and GAPDH examined by semi-quantitative RT-PCR using cDNA prepared from amplified RNA. The first 10 lanes show the expression level of the genes in different bladder cancer patients. The next 2 lanes show the expression level of the genes in bladder from a normal individual; normal transitional cells and bulk. The last 4 lanes show the expression level of the genes in a normal human tissues; Heart, Lung, Liver and Kidney. (b) C2093 and (c) B5860N in tumor cells from 21 bladder cancer patients (1001, 1009, 1010, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 2003, 2014, 3001, 5001, 5002) (upper and middle panel), bladder cancer cell lines (HT1197, UMUC3, J82, HT1376, SW780 and RT4) (lower panel), and normal human tissues (normal bulk; normal bladder, TC; microdissected transitional cells, heart, lung, liver, kidney).

FIG. 2 depicts the results of Northern blot analysis with bladder cancer cell lines and normal human tissues including normal bladder using A0576N(a), C5509(b), F1653(c), B9838(d), C2093(e), B5860N(f), C6055(g,h) DNA fragment as each probe.

FIG. 3 shows Genomic structure of (a) C2093, (b) B5860N and (c) C6055. B5860N has two different variants, designated V1 and V2. C6055 has four different variants, designed MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2.

FIG. 4 depicts the exogenous expression and subcellular localization of C2093, B5860Ns and C6055s. (a) Exogenous expression of C2093 protein by Western blot at 24 and 48 hours after transfection, (b) Subcellular localization of C2093 protein, (c) Cell cycle dependent localization of C2093, (d) Exogenous expression of B5860N V1 (left panel) and B5860N V2 (right panel) proteins by Western blot analysis at 24 and 48 hours after transfection. Subcellular localization of (e) B5860N V1 and (f) B5860N V2 proteins, Cell cycle dependent localization of (g) B5860N V1 proteins, and (h) B5860N V2. (i) Co-transfection with B5860N V1 and B5860N V2 into COST cells. (j) Subcellular localization of C2093 during cell cycle progression. (k) Subcellular localization of B5860N during cell cycle progression. (l) Expression of C6055 protein by Western blot at 36 hours after transfection, (m) Post-translational modification of C6055 protein (n) Subcellular localization of exogenous C6055 protein.

FIG. 5 depicts the growth-inhibitory effects of small-interfering RNAs (siRNAs) designed to reduce the expression of C2093 in bladder cancer cells. (a) Semi-quantitative RT-PCR showing suppression of endogenous expression of C2093 in bladder cancer cell line, UMUC3 cells. GAPDH was used as an internal control. EGFP; EGFP sequence and SCR; scramble sequence as control (see Materials and Methods) (b) Colony-formation assay demonstrating a decrease in the numbers of colonies by knockdown of C2093 in UMUC3 cells. (c) MTT assay demonstrating a decrease in the numbers of colonies by knockdown of C2093 in UMUC3 cells. (d) Semi-quantitative RT-PCR showing suppression of endogenous expression of C2093 in bladder cancer cell line, J82 cells. GAPDH was used as an internal control. (e) Colony-formation assay demonstrating a decrease in the numbers of colonies by knockdown of C2093 in J82 cells. (f) MTT assay demonstrating a decrease in the numbers of colonies by knockdown of C2093 in J82 cells.

FIG. 6 depicts the growth-inhibitory effects of small-interfering RNAs (siRNAs) designed to reduce the expression of B5860N in bladder cancer cells. (a) Semi-quantitative RT-PCR showing suppression of endogenous expression of B5860N in bladder cancer cell line, J82 cells. GAPDH was used as an internal control. EGFP; EGFP sequence and SCR; scramble sequence as controls (see Materials and Methods) (b) Colony-formation assay demonstrating a decrease in the numbers of colonies by knockdown of B5860N in J82 cells. (c) MTT assay demonstrating a decrease in the numbers of colonies by knockdown of B5860N in J82 cells.

FIG. 7 depicts the growth-inhibitory effects of small-interfering RNAs (siRNAs) designed to reduce expression of C6055 in bladder cancer cells. (a) Semi-quantitative RT-PCR showing suppression of endogenous expression of C6055 in bladder cancer cell line, SW780 cells. ACTB was used as an internal control. SCR; scramble sequence as a control (see Materials and Methods) (b) Colony-formation assay demonstrating a decrease in the numbers of colonies by knockdown of C6055 in SW780 cells. (c) MTT assay demonstrating a decrease in the numbers of colonies by knockdown of C6055 in SW780 cells.

FIG. 8 (a) Multi-nucleated cells by treatment of C2093-siRNA. (b) western blotting analysis using anti-C2093 antibody. (c) cell morphology with microscopy.

FIG. 9 (a) Expression of C2093 in bladder cancer tissue sections (right panel ×200; left panel ×100), (b) Expression of B5860N in bladder cancer tissue sections (right panel ×200; left panel ×100), normal bladder tissues (bottom panel).

DETAILED DESCRIPTION OF THE INVENTION

The words “a”, “an” and “the” as used herein mean “at least one” unless otherwise specifically indicated.

Generally bladder cancer cells exist as a solid mass having a highly inflammatory reaction and containing various cellular components. Therefore, previous published microarray data are likely to reflect heterogenous profiles.

With these issues in view, the present inventors prepared purified populations of bladder cancer cells by a method of laser-microbeam microdissection (LMM), and analyzed genome-wide gene-expression profiles of 33 BLCs, using a cDNA microarray representing 27,648 genes. These data not only should provide important information about bladder carcinogenesis, but should also facilitate the identification of candidate genes whose products may serve as diagnostic markers and/or as molecular targets for the treatment of patients with bladder cancer and provide clinically relevant information.

The present invention is based, in part, on the discovery of changes in expression patterns of multiple nucleic acids between epithelial cells and carcinomas of patients with BLC. The differences in gene expression were identified using a comprehensive cDNA microarray system.

The gene-expression profiles of cancer cells from 33 BLCs were analyzed using a cDNA microarray representing 27,648 genes coupled with laser microdissection. By comparing expression patterns between cancer cells from patients diagnosed with BLC and normal ductal epithelial cells purely selected with Laser Microdissection, 394 genes (shown in Table 4) were identified as commonly up-regulated in BLC cells. Similarly, 1272 genes (shown in Table 5) were also identified as being commonly down-regulated in BLC cells. In addition, selection was made of candidate molecular markers having the potential to detect cancer-related proteins in serum or sputum of patients, and some potential targets for development of signal-suppressing strategies in human BLC were discovered. Among them, Tables 4 and 5 provide a list of genes whose expression is altered between BLC and normal tissue.

The differentially expressed genes identified herein find diagnostic utility as markers of BLC and as BLC gene targets, the expression of which may be altered to treat or alleviate a symptom of BLC. The genes whose expression level is modulated (i.e., increased or decreased) in BLC patients are summarized in Tables 4-5 and are collectively referred to herein as “BLC-associated genes”, “BLC nucleic acids” or “BLC polynucleotides” and the corresponding encoded polypeptides are referred to as “BLC polypeptides” or “BLC proteins.” Unless otherwise indicated, the term “BLC” refers to any of the sequences disclosed herein (e.g., BLC-associated genes listed in Tables 4-5). Genes that have been previously described are presented along with a database accession number.

By measuring the expression of the various genes in a sample of cells, BLC can be diagnosed. Similarly, measuring the expression of these genes in response to various agents can identify agents for treating BLC.

The present invention involves determining (e.g., measuring) the expression of at least one, and up to all, of the BLC-associated genes listed in Tables 4-5. Using sequence information provided by the GenBank™ database entries for known sequences, the BLC-associated genes can be detected and measured using techniques well known to one of ordinary skill in the art. For example, sequences within the sequence database entries corresponding to BLC-associated genes can be used to construct probes for detecting RNA sequences corresponding to BLC-associated genes in, e.g., Northern blot hybridization analyses. Probes typically include at least 10, at least 20, at least 50, at least 100, or at least 200 nucleotides of a reference sequence. As another example, the sequences can be used to construct primers for specifically amplifying one or more BLC nucleic acid in, e.g., amplification-based detection methods, such as reverse-transcription based polymerase chain reaction.

Expression level of one or more of BLC-associated gene in a test cell population, e.g., a patient-derived tissues sample, is then compared to the expression level(s) of the same gene(s) in a reference population. The reference cell population includes one or more cells for which the compared parameter is known, i.e., bladder ductal carcinoma cells (e.g., BLC cells) or normal bladder ductal epithelial cells (e.g., non-BLC cells).

Whether or not a pattern of gene expression in a test cell population as compared to a reference cell population indicates BLC or a predisposition thereto depends upon the composition of the reference cell population. For example, if the reference cell population is composed of non-BLC cells, a similarity in gene expression pattern between the test cell population and the reference cell population indicates that the test cell population is non-BLC. Conversely, if the reference cell population is made up of BLC cells, a similarity in gene expression profile between the test cell population and the reference cell population indicates that the test cell population includes BLC cells.

A level of expression of a BLC marker gene in a test cell population is considered “altered” if it varies from the expression level of the corresponding BLC marker gene in a reference cell population by more than 1.1, more than 1.5, more than 2.0, more than 5.0, or more than 10.0 fold.

Differential gene expression between a test cell population and a reference cell population can be normalized to a control nucleic acid, e.g. a housekeeping gene. For example, a control nucleic acid is one which is known not to differ depending on the cancerous or non-cancerous state of the cell. The expression level of a control nucleic acid can be used to normalize signal levels in the test and reference populations. Exemplary control genes include, but are not limited to, e.g., β-actin, glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein P1.

The test cell population can be compared to multiple reference cell populations. Each of the multiple reference populations may differ in the known parameter. Thus, a test cell population may be compared to a first reference cell population known to contain, e.g., BLC cells, as well as a second reference population known to contain, e.g., non-BLC cells (e.g., normal cells). The test cell may be included in a tissue type or cell sample from a subject known to contain, or suspected of containing, BLC cells.

The test cell is preferably obtained from a bodily tissue or a bodily fluid, e.g., biological fluid (such as blood, sputum or urine, for example). For example, the test cell may be purified from bladder tissue. Preferably, the test cell population comprises an epithelial cell. The epithelial cell is preferably from a tissue known to be or suspected to be a bladder ductal carcinoma.

Cells in the reference cell population should be derived from a tissue type similar to that of the test cell. Optionally, the reference cell population is a cell line, e.g. a BLC cell line (i.e., a positive control) or a normal non-BLC cell line (i.e., a negative control). Alternatively, the control cell population may be derived from a database of molecular information derived from cells for which the assayed parameter or condition is known.

The subject is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.

Expression of the genes disclosed herein can be determined at the protein or nucleic acid level, using methods known in the art. For example, Northern hybridization analysis, using probes which specifically recognize one or more of these nucleic acid sequences, can be used to determine gene expression. Alternatively, gene expression may be measured using reverse-transcription-based PCR assays, e.g., using primers specific for the differentially expressed gene sequences. Expression may also be determined at the protein level, i.e., by measuring the level of a polypeptide encoded by a gene described herein, or the biological activity thereof. Such methods are well known in the art and include, but are not limited to, e.g., immunoassays that utilize antibodies to proteins encoded by the genes. The biological activities of the proteins encoded by the genes are generally well known.

To disclose the mechanism of bladder cancer and identify novel diagnostic markers and/or drug targets for the treatment and/or prevention of these tumors, the present inventors analyzed the expression profiles of genes in bladder cancer using a genome-wide cDNA microarray combined with laser microbeam microdissection. As a result, C2093, B5860N and C6055 specifically over-expressed in bladder cancer cells were identified. Furthermore, suppression of the expression of C2093, B5860N or C6055 gene with small interfering RNAs (siRNAs) resulted in a significant growth-inhibition of cancerous cells. These findings suggest that C2093, B5860N and/or C6055 render oncogenic activities to cancer cells, and that inhibition of the activity of one or more of these proteins could be a promising strategy for the treatment and prevention of proliferative diseases such as bladder cancers.

B5860N:

According to the present invention, a cDNA with a similar sequence was identified and encode variants of B5860N. The cDNA of the longer variant consists of 5318 nucleotides and contains an open reading frame of 2436 nucleotides (SEQ ID NO: 3). The open reading frame of known B5860N consists of 1584 nucleotide and encodes a 527 amino acid-protein (GeneBank Accession Number NM_(—)017779). Therefore, the longer variant, consisting of 5318 nucleotide, is novel to the instant invention. Furthermore, the known sequence of the B5860N cDNA encoding the 527 amino acid-protein consists of 3338 nucleotides. However, in the present invention, a full length cDNA of B5860N consisting of 4466 nucleotide was isolated. The nucleotide sequence of this shorter variant comprises a novel sequence of 3′-UTR as compared with the known nucleotide sequence, although both of the amino acid sequences encoded thereby were identical. In the present specification, the transcripts of the shorter variant, encoding the known 527 amino acid-protein, and the longer variant, encoding the novel 811 amino acid-protein, are described herein as B5860NV2 and B5860NV1, respectively. The nucleotide sequence of B5860NV1 and B5860NV2, and amino acid sequence encoded thereby are set forth in the following SEQ ID NOs.

nucleotide amino acid sequence sequence B5860NV1 SEQ ID NO: 3 SEQ ID NO: 4 B5860NV2 SEQ ID NO: 5 SEQ ID NO: 6

Thus, the present invention provides substantially pure polypeptides encoded by the longer variant B5860NV1, including polypeptides comprising the amino acid sequence of SEQ ID NO: 4, as well as functional equivalents thereof, to the extent that they encode a B5860NV1 protein. Examples of polypeptides functionally equivalent to B5860NV1 include, for example, homologous proteins of other organisms corresponding to the human B5860NV1 protein, as well as mutants of human B5860NV1 proteins.

C6055:

According to the present invention, a cDNA with a similar sequence was identified and encode variants of C6055. According to the database from NCBI, C6055 consists of 24 exons, designated MGC34032, located on the chromosome 1p31.3. Because C6055 is not included within last exon (exon24) of MGC34032 on database, we performed RT-PCR as EST-walking, and 5′RACE and 3′RACE experiments using bladder cancer cell line, SW780, as a template to obtain the entire cDNA sequence of C6055 (see Materials and Methods). As a result, we found two novel transcripts, C6055V1 and C6055V2. Eventually, this gene has four different splicing variants consisting of 24, 25, 22 and 22 exons, corresponding to MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2, respectively (FIG. 3 c). There were alternative splicing in exon 1, 2, 3, 4 and 24 of MGC34032, and the other remaining exons were common among four transcripts. C6055V1 and C6055V2 transcripts have no exon 1, 2 and 3 of MGC34032, generating same stop codon within last exon. In particular, the ORF of C6055V1 and C6055V2 transcripts start at within each exon 4, indicating C6055V1 and C6055V2 transcripts have same ORF. The full-length cDNA sequences of MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2 transcripts consist of 2302, 3947, 3851, and 3819 nucleotides, respectively. Eventually, MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2 transcripts encode 719, 587, 675 and 675 amino acids, respectively. The nucleotide sequence of C6055V1 and C6055V2, and amino acid sequence encoded thereby are set forth in the following SEQ ID NOs.

nucleotide amino acid sequence sequence C6055V1 SEQ ID NO: 129 SEQ ID NO: 130 C6055V2 SEQ ID NO: 131 SEQ ID NO: 132

Thus, the present invention provides substantially pure polypeptides encoded by the longer variant C6055V1 or C6055V2, including polypeptides comprising the amino acid sequence of SEQ ID NO: 130 or SEQ ID NO: 132, as well as functional equivalents thereof, to the extent that they encode a Genbank Accession No. AK128063 protein. Examples of polypeptides functionally equivalent to C6055V1 or C6055V2 include, for example, homologous proteins of other organisms corresponding to the human C6055V1 or C6055V2 protein, as well as mutants of human C6055V1 or C6055V2 proteins.

In the present invention, the term “functionally equivalent” means that the subject polypeptide has the activity to promote cell proliferation like the B5860NV1 protein and to confer oncogenic activity to cancer cells. Whether the subject polypeptide has a cell proliferation activity or not can be judged by introducing the DNA encoding the subject polypeptide into a cell, expressing the respective polypeptide and detecting promotion of proliferation of the cells or increase in colony forming activity. Such cells include, for example, NIH3T3, COS7 and HEK293.

Methods for preparing polypeptides functionally equivalent to a given protein are well known by a person skilled in the art and include known methods of introducing mutations into the protein. For example, one skilled in the art can prepare polypeptides functionally equivalent to the human B5860NV1 protein by introducing an appropriate mutation in the amino acid sequence of this protein by site-directed mutagenesis (Hashimoto-Gotoh et al., (1995) Gene 152:271-5; Zoller and Smith, (1983) Methods Enzymol 100: 468-500; Kramer et al., (1984) Nucleic Acids Res. 12:9441-56; Kramer and Fritz, (1987) Methods Enzymol 154: 350-67; Kunkel, (1985) Proc Natl Acad Sci USA 82: 488-92; Kunkel, (1991) Methods Enzymol; 204:125-39). Amino acid mutations can occur in nature, too. The polypeptide of the present invention includes those proteins having the amino acid sequences of the human B5860NV1 protein in which one or more amino acids are mutated, provided the resulting mutated polypeptides are functionally equivalent to the human B5860NV1 protein. In the present invention, the number of mutation is generally no more than 35%, preferably no more than 30%, even more preferably no more than 25%, 20%, 10%, 5%, 2% or 1% of all amino acids. Specifically, the number of amino acids to be mutated in such a mutant is generally 200 or 100 amino acids or less, typically 10 amino acids or less, preferably 6 amino acids or less, and more preferably 3 amino acids or less.

Mutated or modified proteins, proteins having amino acid sequences modified by substituting, deleting, inserting and/or adding one or more amino acid residues of a certain amino acid sequence, have been known to retain the original biological activity (Mark et al., (1984) Proc Natl Acad Sci USA 81: 5662-6; Zoller and Smith, (1982) Nucleic Acids Res 10:6487-500; Dalbadie-McFarland et al., (1982) Proc Natl Acad Sci USA 79: 6409-13).

To that end, the amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Note, the parenthetic letters indicate the one-letter codes of amino acids.

An example of a polypeptide in which one or more amino acids residues are added to the amino acid sequence of human B5860NV1 protein is a fusion protein containing the human B5860NV1 protein. Fusion proteins, fusions of the human B5860NV1 protein and other peptides or proteins, are included in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, such as by linking the DNA encoding the human B5860NV1 protein of the invention with DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins that may be fused to the protein of the present invention.

Known peptides that can be used as peptides that are fused to a protein of the present invention include, for example, FLAG (Hopp et al., (1988) Biotechnology 6: 1204-10), 6×His containing six His (histidine) residues, 10×His, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment and the like. Examples of proteins that may be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP (maltose-binding protein) and such.

Fusion proteins can be prepared by fusing commercially available DNA, encoding the fusion peptides or proteins discussed above, with a DNA encoding a polypeptide of the present invention and expressing the fused DNA prepared.

Alternatively, functionally equivalent polypeptides may be isolated using methods known in the art, for example, using a hybridization technique (Sambrook et al., (1989) Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press). One skilled in the art can readily isolate a DNA having high homology with a whole or part of the DNA sequence encoding the human B5860NV1 protein (i.e., SEQ ID NO: 3), and isolate functionally equivalent polypeptides to the human B5860NV1 protein from the isolated DNA. The polypeptides of the present invention include those that are encoded by DNA that hybridize with a whole or part of the DNA sequence encoding the human B5860NV1 protein and are functionally equivalent to the human B5860NV1 protein. These polypeptides include mammalian homologues corresponding to the human-derived protein (for example, a polypeptide encoded by a monkey, rat, rabbit and bovine gene). In isolating a cDNA highly homologous to the DNA encoding the human B5860NV1 protein from animals, it is particularly preferable to use tissues from testis or bladder cancer tissue.

The condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the human B5860NV1 protein can be routinely selected by a person skilled in the art. For example, hybridization may be performed by conducting pre-hybridization at 68° C. for 30 min or longer using “Rapid-hyb buffer” (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68° C. for 1 hour or longer. The following washing step can be conducted, for example, in a low stringency condition. A low stringency condition is, for example, 42° C., 2×SSC, 0.1% SDS, or preferably 50° C., 2×SSC, 0.1% SDS. More preferably, high stringency conditions are used. A high stringency condition is, for example, washing 3 times in 2×SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1×SSC, 0.1% SDS at 37° C. for 20 min, and washing twice in 1×SSC, 0.1% SDS at 50° C. for 20 min. However, several factors, such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.

In place of hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a polypeptide functionally equivalent to the human B5860NV1 protein, using a primer synthesized based on the sequence information of the protein encoding DNA (SEQ ID NO: 3).

Polypeptides that are functionally equivalent to the human B5860NV1 protein, encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques, normally have a high homology to the amino acid sequence of the human B5860NV1 protein. As used herein, the term “high homology” typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 85%, 90%, 93%, 95%, 98%, 99% or higher between a polypeptide sequence or a polynucleotide sequence and a reference sequence. Percent homology (also referred to as percent identity) is typically determined between two optimally aligned sequences. Methods of aligning sequences for comparison are well-known in the art. Optimal alignment of sequences and comparison can be conducted, e.g., using the algorithm in “Wilbur and Lipman, (1983) Proc Natl Acad Sci USA 80: 726-30”.

A polypeptide of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a function equivalent to that of the human B5860NV1 protein of the present invention, it is within the scope of the present invention.

The polypeptides of the present invention can be prepared as recombinant proteins or natural proteins, using methods well known to those skilled in the art. A recombinant protein can be prepared, for example, by inserting a DNA, which encodes a polypeptide of the present invention (for example, the DNA comprising the nucleotide sequence of SEQ ID NO: 3), into an appropriate expression vector, introducing the vector into an appropriate host cell, obtaining the extract, and purifying the polypeptide by subjecting the extract to chromatography, e.g., ion exchange chromatography, reverse phase chromatography, gel filtration or affinity chromatography utilizing a column to which antibodies against the protein of the present invention is fixed or by combining more than one of aforementioned columns.

In addition, when the polypeptide of the present invention is expressed within host cells (for example, animal cells and E. coli) as a fusion protein with glutathione-S-transferase protein or as a recombinant protein supplemented with multiple histidines, the expressed recombinant protein can be purified using a glutathione column or nickel column. Alternatively, when the polypeptide of the present invention is expressed as a protein tagged with c-myc, multiple histidines or FLAG, it can be detected and purified using antibodies to c-myc, His or FLAG, respectively.

After purifying the fusion protein, it is also possible to exclude regions other than the objective polypeptide by cutting the fusion protein with thrombin or factor-Xa as required.

A natural protein can be isolated by methods known to a person skilled in the art, for example, by contacting the affinity column, in which antibodies binding to the B5860NV1 protein described below are bound, with the extract of tissues or cells expressing the polypeptide of the present invention. The antibodies can be polyclonal antibodies or monoclonal antibodies.

The present invention also encompasses partial peptides of the polypeptides of the present invention. Preferably, the partial peptides of the present invention comprise an amino acid sequence selected from positions 304 to 588 of the amino acid sequence of SEQ ID NO: 4, or a part thereof. The amino acid sequence extending between positions 304 and 588 is a B5860NV1-specific region, as compared to B5860NV2. The partial peptide has an amino acid sequence specific to the polypeptide of the present invention and consists of at least 7 amino acids, preferably 8 amino acids or more, and more preferably 9 amino acids or more. The partial peptide can be used, for example, for preparing antibodies against the polypeptide of the present invention, screening for a compound that binds to the polypeptide of the present invention, and screening for inhibitors of the polypeptide of the present invention.

A partial peptide of the invention can be produced by genetic engineering, by known methods of peptide synthesis or by digesting the polypeptide of the invention with an appropriate peptidase. For peptide synthesis, for example, solid phase synthesis or liquid phase synthesis may be used.

The present invention further provides polynucleotides that encode such B5860NV1 polypeptides described above. The polynucleotides of the present invention can be used for the in vivo or in vitro production of a polypeptide of the present invention as described above, or can be applied to gene therapy for diseases attributed to genetic abnormality in the gene encoding the protein of the present invention. Any form of the polynucleotide of the present invention can be used, so long as it encodes a polypeptide of the present invention, including mRNA, RNA, cDNA, genomic DNA, chemically synthesized polynucleotides. The polynucleotide of the present invention includes a DNA comprising a given nucleotide sequences as well as its degenerate sequences, so long as the resulting DNA encodes a polypeptide of the present invention.

A polynucleotide of the present invention can be prepared by methods known to a person skilled in the art. For example, a polynucleotide of the present invention can be prepared by: preparing a cDNA library from cells which express a polypeptide of the present invention, and conducting hybridization using a partial sequence of the DNA of the present invention (for example, SEQ ID NO: 3) as a probe. A cDNA library can be prepared, for example, by the method described in Sambrook et al., (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press; alternatively, commercially available cDNA libraries may be used. A cDNA library can be also prepared by: extracting RNAs from cells expressing the polypeptide of the present invention, synthesizing oligo DNAs based on the sequence of the DNA of the present invention (for example, SEQ ID NO: 3), conducting PCR using the oligo DNAs as primers, and amplifying cDNAs encoding the protein of the present invention.

In addition, by sequencing the nucleotides of the obtained cDNA, the translation region encoded by the cDNA can be routinely determined, and the amino acid sequence of the polypeptide of the present invention can be easily obtained. Moreover, by screening the genomic DNA library using the obtained cDNA or parts thereof as a probe, the genomic DNA can be isolated.

More specifically, mRNAs may first be prepared from a cell, tissue or organ (e.g., testis) or bladder cancer cell line in which the object polypeptide of the invention is expressed. Known methods can be used to isolate mRNAs; for instance, total RNA may be prepared by guanidine ultracentrifugation (Chirgwin et al., (1979) Biochemistry 18:5294-9) or AGPC method (Chomczynski and Sacchi, (1987) Anal Biochem 162:156-9). In addition, mRNA may be purified from total RNA using mRNA Purification Kit (Pharmacia) and such. Alternatively, mRNA may be directly purified by QuickPrep mRNA Purification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reverse transcriptase. cDNA may be synthesized using a commercially available kit, such as the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo). Alternatively, cDNA may be synthesized and amplified following the 5′-RACE method (Frohman et al., (1988) Proc Natl Acad Sci USA 85: 8998-9002; Belyaysky et al., (1989) Nucleic Acids Res 17: 2919-32), which uses a primer and such, described herein, the 5′-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction (PCR).

A desired DNA fragment is prepared from the PCR products and ligated with a vector DNA. The recombinant vectors are used to transform E. coli and such, and a desired recombinant vector is prepared from a selected colony. The nucleotide sequence of the desired DNA can be verified by conventional methods, such as dideoxynucleotide chain termination.

The nucleotide sequence of a polynucleotide of the invention may be designed to be expressed more efficiently by taking into account the frequency of codon usage in the host to be used for expression (Grantham et al., (1981) Nucleic Acids Res 9: 43-74). The sequence of the polynucleotide of the present invention may be altered by a commercially available kit or a conventional method. For instance, the sequence may be altered by digestion with restriction enzymes, insertion of a synthetic oligonucleotide or an appropriate polynucleotide fragment, addition of a linker, or insertion of the initiation codon (ATG) and/or the stop codon (TAA, TGA or TAG).

Specifically, the polynucleotide of the present invention encompasses the DNA comprising the nucleotide sequence of SEQ ID NO: 3.

Furthermore, the present invention provides a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a nucleotide sequence of SEQ ID NO: 3, and encodes a polypeptide functionally equivalent to the B5860NV1 protein of the invention described above. One skilled in the art may appropriately choose the appropriately stringent conditions. For example, low stringency condition can be used. More preferably, high stringency condition can be used. These conditions are the same as that described above. The hybridizing DNA above is preferably a cDNA or a chromosomal DNA.

The present invention also provides a polynucleotide which is complementary to the polynucleotide encoding human B5860NV1 protein (SEQ ID NO: 3) or the complementary strand thereof, and which comprises at least 15 nucleotides, wherein the polynucleotide hybridizes with the nucleotide sequence extending between positions 988 and 1842 of SEQ ID NO:3. The polynucleotide of the present invention is preferably a polynucleotide which specifically hybridizes with the DNA encoding the B5860NV1 polypeptide of the present invention. The term “specifically hybridize” as used herein, means that significant cross-hybridization does not occur with DNA encoding other proteins, under the usual hybridizing conditions, preferably under stringent hybridizing conditions. Such polynucleotides include, probes, primers, nucleotides and nucleotide derivatives (for example, antisense oligonucleotides and ribozymes), which specifically hybridize with DNA encoding the polypeptide of the invention or its complementary strand. Moreover, such polynucleotide can be utilized for the preparation of DNA chip.

Vectors and Host Cells

The present invention also provides a vector and host cell into which a polynucleotide of the present invention is introduced. A vector of the present invention is useful to keep a polynucleotide, especially a DNA, of the present invention in host cell, to express the polypeptide of the present invention, or to administer the polynucleotide of the present invention for gene therapy.

When E. coli is the host cell and the vector is amplified and produced in a large amount in E. coli (e.g., JM109, DH5α, HB101 or XL1Blue), the vector should have “ori” to be amplified in E. coli and a marker gene for selecting transformed E. coli (e.g., a drug-resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol or the like). For example, the M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, etc. can be used. In addition, pGEM-T, pDIRECT and pT7 can also be used for subcloning and extracting cDNA as well as the vectors described above. When a vector is used to produce a protein of the present invention, an expression vector is especially useful. For example, an expression vector to be expressed in E. coli should have the above characteristics to be amplified in E. coli. When E. coli, such as JM109, DH5α, HB101 or XL1 Blue, are used as a host cell, the vector should have a promoter, for example, lacZ promoter (Ward et al., (1989) Nature 341: 544-6; (1992) FASEB J 6: 2422-7), araB promoter (Better et al., (1988) Science 240: 1041-3), T7 promoter or the like, that can efficiently express the desired gene in E. coli. In that respect, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP and pET (in this case, the host is preferably BL21 which expresses T7 RNA polymerase), for example, can be used instead of the above vectors. Additionally, the vector may also contain a signal sequence for polypeptide secretion. An exemplary signal sequence that directs the polypeptide to be secreted to the periplasm of the E. coli is the pelB signal sequence (Lei et al., (1987) J Bacteriol 169: 4379-83.). Means for introducing of the vectors into the target host cells include, for example, the calcium chloride method, and the electroporation method.

In addition to E. coli, for example, expression vectors derived from mammals (for example, pcDNA3 (Invitrogen) and pEF-BOS (Mizushima S and Nagata S, (1990) Nucleic Acids Res 18 (17): 5322), pEF, pCDM8), expression vectors derived from insect cells (for example, “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g., pZIpneo), expression vector derived from yeast (e.g., “Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01) and expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50) can be used for producing the polypeptide of the present invention.

In order to express the vector in animal cells, such as CHO, COS or NIH3T3 cells, the vector should have a promoter necessary for expression in such cells, for example, the SV40 promoter (Mulligan et al., (1979) Nature 277: 108), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., (1990) Nucleic Acids Res 18: 5322), the CMV promoter and the like, and preferably a marker gene for selecting transformants (for example, a drug resistance gene selected by a drug (e.g., neomycin, G418)). Examples of known vectors with these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

Producing Polypeptides

In addition, the present invention provides methods for producing a polypeptide of the present invention. The polypeptides may be prepared by culturing a host cell which harbors an expression vector comprising a gene encoding the polypeptide. According to needs, methods may be used to express a gene stably and, at the same time, to amplify the copy number of the gene in cells. For example, a vector comprising the complementary DHFR gene (e.g., pCHO I) may be introduced into CHO cells in which the nucleic acid synthesizing pathway is deleted, and then amplified by methotrexate (MTX). Furthermore, in case of transient expression of a gene, the method wherein a vector comprising a replication origin of SV40 (pcD, etc.) is transformed into COS cells comprising the SV40 T antigen expressing gene on the chromosome can be used.

A polypeptide of the present invention obtained as above may be isolated from inside or outside (such as medium) of host cells and purified as a substantially pure homogeneous polypeptide. The term “substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The method for polypeptide isolation and purification is not limited to any specific method; in fact, any standard method may be used.

For instance, column chromatography, filter, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization may be appropriately selected and combined to isolate and purify the polypeptide.

Examples of chromatography include, for example, affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and such (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., (1996) Cold Spring Harbor Laboratory Press). These chromatographies may be performed by liquid chromatography, such as HPLC and FPLC. Thus, the present invention provides for highly purified polypeptides prepared by the above methods.

A polypeptide of the present invention may be optionally modified or partially deleted by treating it with an appropriate protein modification enzyme before and/or after purification. Useful protein modification enzymes include, but are not limited to, trypsin, chymotrypsin, lysylendopeptidase, protein kinase, glucosidase and so on.

Diagnosing Bladder Cancer:

In the context of the present invention, BLC is diagnosed by measuring the expression level of one or more BLC nucleic acids from a test population of cells, (i.e., a patient-derived biological sample). Preferably, the test cell population contains an epithelial cell, e.g., a cell obtained from bladder tissue. Gene expression can also be measured from blood or other bodily fluids such as urine. Other biological samples can be used for measuring protein levels. For example, the protein level in blood or serum derived from a subject to be diagnosed can be measured by immunoassay or other conventional biological assay.

Expression of one or more BLC-associated genes, e.g., genes listed in Tables 4-5, is determined in the test cell or biological sample and compared to the normal control expression level associated with the one or more BLC-associated gene(s) assayed. A normal control level is an expression profile of a BLC-associated gene typically found in a population known not to be suffering from BLC. An alteration (e.g., an increase or decrease) in the level of expression in the patient-derived tissue sample of one or more BLC-associated genes indicates that the subject is suffering from or is at risk of developing BLC. For example, an increase in the expression of one or more up-regulated BLC-associated genes listed in Table 4 in the test population as compared to the normal control level indicates that the subject is suffering from or is at risk of developing BLC. Conversely, a decrease in expression of one or more down-regulated BLC-associated genes listed in Table 5 in the test population as compared to the normal control level indicates that the subject is suffering from or is at risk of developing BLC.

Alteration of one or more of the BLC-associated genes in the test population as compared to the normal control level indicates that the subject suffers from or is at risk of developing BLC. For example, alteration of at least 1%, at least 5%, at least 25%, at least 50%, at least 60%, at least 80%, or at least 90% or more of the panel of BLC-associated genes (genes listed in Tables 4-5) indicates that the subject suffers from or is at risk of developing BLC.

Moreover, the present invention provides a method for diagnosing cell proliferative disease such as bladder cancer using the expression level of the genes of the present invention as a diagnostic marker. This diagnostic method comprises the steps of: (a) detecting the expression level of one or more of C2093, B5860Ns and C6055s gene; and (b) relating an elevation of the expression level to bladder cancer. In the context of the present invention, the transcript of the B5860N gene includes B5860NV1 and B5860NV2. In the context of the present invention, the transcript of the C6055 gene includes MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2.

The expression levels of the C2093, B5860Ns or C6055s gene in a biological sample can be estimated by quantifying mRNA corresponding to or protein encoded by the C2093, B5860Ns or C6055s gene. Quantification methods for mRNA are known to those skilled in the art. For example, the levels of mRNAs corresponding to the C2093, B5860Ns or C6055s gene can be estimated by Northern blotting or RT-PCR. Since the full-length nucleotide sequences of the C2093 gene is shown in SEQ ID NO: 1. Alternatively, the full-length nucleotide sequences of two variant forms of B5860N gene transcripts are also shown in SEQ ID NO: 3 and 5. Alternatively, the full-length nucleotide sequences of four variant forms of C6055 gene transcripts are also shown in SEQ ID NO: 129, 131, 133 and 135. Accordingly, anyone skilled in the art can design the nucleotide sequences for probes or primers to quantify the C2093, B5860N or C6055 gene.

Also, the expression level of the C2093, B5860Ns or C6055s gene can be analyzed based on the activity or quantity of protein encoded by the gene. A method for determining the quantity of the C2093, B5860N or C6055 protein is shown in below. For example, immunoassay methods are useful for the determination of the proteins in biological materials. Any biological materials can be used as the biological sample for the determination of the protein or its activity, so long as the marker gene (i.e, the C2093, B5860Ns or C6055s gene) is expressed in the sample of a bladder cancer patient. For example, in the context of the present invention, bladder tissue is a preferred biological sample. However, bodily fluids, such as blood and urine, may be also analyzed. On the other hand, a suitable method can be selected for the determination of the activity of a protein encoded by the C2093, B5860Ns or C6055s gene according to the activity of a protein to be analyzed.

Expression levels of the C2093, B5860Ns or C6055s gene in a biological sample are estimated and compared with those in a normal sample (e.g., a sample derived from a non-diseased subject). When such a comparison shows that the expression level of the target gene is higher than those in the normal sample, the subject is judged to be affected with bladder cancer. The expression level of the C2093, B5860Ns or C6055s gene in the biological samples from a normal subject and subject to be diagnosed may be determined at the same time. Alternatively, normal ranges of the expression levels can be determined by a statistical method based on the results obtained by analyzing the expression level of the gene in samples previously collected from a control group. A result obtained by comparing the sample of a subject is compared with the normal range; when the result does not fall within the normal range, the subject is judged to be affected with or is at risk of developing bladder cancer.

In the present invention, a diagnostic agent for diagnosing cell proliferative disease, such as bladder cancer, is also provided. The diagnostic agent of the present invention comprises a compound that binds to C2093, B5860Ns or C6055s gene transcript or polypeptide encoded thereby. Preferably, an oligonucleotide that hybridizes to the polynucleotide comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 129, 131, 133 and 135, or an antibody that binds to the polypeptide consisting of amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 130, 132, 134 and 136 may be used as such a compound.

Identifying Agents that Inhibit or Enhance BLC-Associated Gene Expression:

An agent that inhibits the expression of a BLC-associated gene or the activity of its gene product can be identified by contacting a test cell population expressing a BLC-associated up-regulated gene with a test agent and then determining the expression level of the BLC-associated gene or the activity of its gene product. A decrease in the level of expression of the BLC-associated gene or in the level of activity of its gene product in the presence of the agent as compared to the expression or activity level in the absence of the test agent indicates that the agent is an inhibitor of a BLC-associated up-regulated gene and useful in inhibiting BLC.

Alternatively, an agent that enhances the expression of a BLC-associated down-regulated gene or the activity of its gene product can be identified by contacting a test cell population expressing a BLC-associated gene with a test agent and then determining the expression level or activity of the BLC-associated down-regulated gene. An increase in the level of expression of the BLC-associated gene or in the level of activity of its gene product as compared to the expression or activity level in the absence of the test agent indicates that the test agent augments expression of the BLC-associated down-regulated gene or the activity of its gene product.

The test cell population may be any cell expressing the BLC-associated genes. For example, the test cell population may contain an epithelial cell, such as a cell derived from bladder tissue. Furthermore, the test cell may be an immortalized cell line derived from a carcinoma cell. Alternatively, the test cell may be a cell which has been transfected with a BLC-associated gene or which has been transfected with a regulatory sequence (e.g., a promoter sequence) from a BLC-associated gene operably linked to a reporter gene.

Assessing Efficacy of Treatment of BLC in a Subject:

The differentially expressed BLC-associated genes identified herein also allow for the course of treatment of BLC to be monitored. In this method, a test cell population is provided from a subject undergoing treatment for BLC. If desired, test cell populations are obtained from the subject at various time points, for example, before, during, and/or after treatment. Expression of one or more of the BLC-associated genes in the cell population is then determined and compared to a reference cell population which includes cells whose BLC state is known. In the context of the present invention, the reference cells should have not been exposed to the treatment of interest.

If the reference cell population contains no BLC cells, a similarity in the expression of a BLC-associated gene in the test cell population and the reference cell population indicates that the treatment of interest is efficacious. However, a difference in the expression of a BLC-associated gene in the test population and a normal control reference cell population indicates a less favorable clinical outcome or prognosis. Similarly, if the reference cell population contains BLC cells, a difference between the expression of a BLC-associated gene in the test cell population and the reference cell population indicates that the treatment of interest is efficacious, while a similarity in the expression of a BLC-associated gene in the test population and a bladder cancer control reference cell population indicates a less favorable clinical outcome or prognosis.

Additionally, the expression level of one or more BLC-associated genes determined in a subject-derived biological sample obtained after treatment (i.e., post-treatment levels) can be compared to the expression level of the one or more BLC-associated genes determined in a subject-derived biological sample obtained prior to treatment onset (i.e., pre-treatment levels). If the BLC-associated gene is an up-regulated gene, a decrease in the expression level in a post-treatment sample indicates that the treatment of interest is efficacious while an increase or maintenance in the expression level in the post-treatment sample indicates a less favorable clinical outcome or prognosis. Conversely, if the BLC-associated gene is an down-regulated gene, an increase in the expression level in a post-treatment sample may indicate that the treatment of interest is efficacious while an decrease or maintenance in the expression level in the post-treatment sample indicates a less favorable clinical outcome or prognosis.

As used herein, the term “efficacious” indicates that the treatment leads to a reduction in the expression of a pathologically up-regulated gene, an increase in the expression of a pathologically down-regulated gene or a decrease in size, prevalence, or metastatic potential of bladder ductal carcinoma in a subject. When a treatment of interest is applied prophylactically, the term “efficacious” means that the treatment retards or prevents a bladder tumor from forming or retards, prevents, or alleviates a symptom of clinical BLC. Assessment of bladder tumors can be made using standard clinical protocols. In addition, efficaciousness can be determined in association with any known method for diagnosing or treating BLC. BLC can be diagnosed, for example, by identifying symptomatic anomalies, e.g., weight loss, abdominal pain, back pain, anorexia, nausea, vomiting and generalized malaise, weakness, and jaundice.

The present method of diagnosing bladder cancer may be applied for assessing the efficacy of treatment of bladder cancer in a subject. According to the method, a biological sample, such as a test cell population, is obtained from a subject undergoing treatment for bladder cancer. The method for assessment can be conducted according to conventional methods of diagnosing bladder cancer.

If desired, biological samples are obtained from the subject at various time points before, during or after the treatment. The expression level of the C2093, B5860Ns or C6055s gene, in the biological sample is then determined and compared to a control level derived, for example, from a reference cell population which includes cells whose state of bladder cancer (i.e., cancerous cell or non-cancerous cell) is known. The control level is determined in a biological sample that has not been exposed to the treatment. If the control level is derived from a biological sample which contains no cancerous cell, a similarity between the expression level in the subject-derived biological sample and the control level indicates that the treatment is efficacious. A difference between the expression level of the C2093, B5860Ns or C6055s gene in the subject-derived biological sample and the control level indicates a less favorable clinical outcome or prognosis.

The term “efficacious” refers that the treatment leads to a reduction in the expression of a pathologically up-regulated gene (e.g., the C2093, B5860Ns and C6055s gene) or a decrease in size, prevalence or proliferating potential of bladder cancer cells in a subject. When a treatment is applied prophylactically, “efficacious” indicates that the treatment retards or prevents occurrence of bladder cancer. The assessment of bladder cancer can be made using standard clinical protocols. Furthermore, the efficaciousness of a treatment may be determined in association with any known method for diagnosing or treating bladder cancer. Moreover, the present method of diagnosing bladder cancer may also be applied for assessing the prognosis of a subject with bladder cancer by comparing the expression level of the C2093, B5860Ns or C6055s gene in a patient-derived biological sample, such as test cell population, to a control level. Alternatively, the expression level of the C2093, B5860Ns or C6055s gene in a biological sample derived from patients may be measured over a spectrum of disease stages to assess the prognosis of the patient.

An increase in the expression level of the C2093, B5860Ns or C6055s gene as compared to a normal control level indicates less favorable prognosis. A similarity in the expression level of the C2093, B5860Ns or C6055s gene compared to a normal control level indicates a more favorable prognosis for the patient.

Selecting a Therapeutic Agent for Treating BLC that is Appropriate for a Particular Individual:

Differences in the genetic makeup of individuals can result in differences in their relative abilities to metabolize various drugs. An agent that is metabolized in a subject to act as an anti-BLC agent can manifest itself by inducing a change in a gene expression pattern in the subject's cells from that characteristic of a cancerous state to a gene expression pattern characteristic of a non-cancerous state. Accordingly, the differentially expressed BLC-associated genes disclosed herein allow for a putative therapeutic or prophylactic inhibitor of BLC to be tested in a test cell population from a selected subject in order to determine if the agent is a suitable inhibitor of BLC in the subject.

To identify an inhibitor of BLC that is appropriate for a specific subject, a test cell population from the subject is exposed to a therapeutic agent, and the expression of one or more of BLC-associated genes listed in Table 4-5 is determined.

In the context of the method of the present invention, the test cell population contains a BLC cell expressing a BLC-associated gene. Preferably, the test cell is an epithelial cell. For example, a test cell population may be incubated in the presence of a candidate agent and the pattern of gene expression of the test cell population may be measured and compared to one or more reference profiles, e.g., a BLC reference expression profile or a non-BLC reference expression profile.

A decrease in expression of one or more of the BLC-associated genes listed in Table 4 or an increase in expression of one or more of the BLC-associated genes listed in Table 5 in a test cell population relative to a reference cell population containing BLC indicates that the agent has therapeutic potential.

In the context of the present invention, the test agent can be any compound or composition. Exemplary test agents include, but are not limited to, immunomodulatory agents.

Screening Assays for Identifying Therapeutic Agents:

The differentially expressed BLC-associated genes disclosed herein can also be used to identify candidate therapeutic agents for treating BLC. The method of the present invention involves screening a candidate therapeutic agent to determine if it can convert an expression profile of one or more BLC-associated genes listed in Tables 4-5 characteristic of a BLC state to a gene expression pattern characteristic of a non-BLC state.

In the instant method, a cell is exposed to a test agent or a plurality of test agents (sequentially or in combination) and the expression of one or more of the BLC-associated genes listed in Tables 4-5 in the cell is measured. The expression profile of the BLC-associated gene(s) assayed in the test population is compared to expression level of the same BLC-associated gene(s) in a reference cell population that is not exposed to the test agent.

An agent capable of stimulating the expression of an under-expressed gene or suppressing the expression of an over-expressed genes has potential clinical benefit. Such agents may be further tested for the ability to prevent bladder ductal carcinomal growth in animals or test subjects.

In a further embodiment, the present invention provides methods for screening candidate agents which act on the potential targets in the treatment of BLC. As discussed in detail above, by controlling the expression levels of marker genes or the activities of their gene products, one can control the onset and progression of BLC. Thus, candidate agents, which act on the potential targets in the treatment of BLC, can be identified through screening methods that use such expression levels and activities as indices of the cancerous or non-cancerous state. In the context of the present invention, such screening may comprise, for example, the following steps:

-   -   a) contacting a test compound with a polypeptide encoded by a         polynucleotide selected from the group consisting of the genes         listed in Table 4 or 5;     -   b) detecting the binding activity between the polypeptide and         the test compound; and     -   c) selecting the test compound that binds to the polypeptide.

Alternatively, the screening method of the present invention may comprise the following steps:

-   -   a) contacting a candidate compound with a cell expressing one or         more marker genes, wherein the one or more marker genes are         selected from the group consisting of the genes listed in Table         4 or 5; and     -   b) selecting the candidate compound that reduces the expression         level of one or more marker genes selected from the group         consisting of the genes listed in Table 4, or elevates the         expression level of one or more marker genes selected from the         group consisting of the genes listed in Table 5.

Cells expressing a marker gene include, for example, cell lines established from BLC; such cells can be used for the above screening of the present invention.

Alternatively, the screening method of the present invention may comprise the following steps:

-   -   a) contacting a test compound with a polypeptide encoded by a         polynucleotide selected from the group consisting of the genes         listed in Table 4 or 5;     -   b) detecting the biological activity of the polypeptide of step         (a); and     -   c) selecting a compound that suppresses the biological activity         of the polypeptide encoded by the polynucleotide selected from         the group consisting of the genes listed in Table 4 as compared         to the biological activity detected in the absence of the test         compound, or enhances the biological activity of the polypeptide         encoded by the polynucleotide selected from the group consisting         of the genes listed in Table 5 as compared to the biological         activity detected in the absence of the test compound.

A protein for use in the screening method of the present invention can be obtained as a recombinant protein using the nucleotide sequence of the marker gene. Based on the information regarding the marker gene and its encoded protein, one skilled in the art can select any biological activity of the protein as an index for screening and any suitable measurement method to assay for the selected biological activity.

Alternatively, the screening method of the present invention may comprise the following steps:

-   -   a) contacting a candidate compound with a cell into which a         vector, comprising the transcriptional regulatory region of one         or more marker genes and a reporter gene that is expressed under         the control of the transcriptional regulatory region, has been         introduced, wherein the one or more marker genes are selected         from the group consisting of the genes listed in Table 4 or 5;     -   b) measuring the expression or activity of said reporter gene;         and     -   c) selecting the candidate compound that reduces the expression         level or activity of said reporter gene when said marker gene is         an up-regulated marker gene selected from the group consisting         of the genes listed in Table 4, or that enhances the expression         level or activity of said reporter gene when said marker gene is         a down-regulated marker gene selected from the group consisting         of the genes listed in Table 5, as compared to the expression         level or activity detected in the absence of the test compound.

Suitable reporter genes and host cells are well known in the art. A reporter construct suitable for the screening method of the present invention can be prepared by using the transcriptional regulatory region of a marker gene. When the transcriptional regulatory region of the marker gene is known to those skilled in the art, a reporter construct can be prepared by using the previous sequence information. When the transcriptional regulatory region of the marker gene remains unidentified, a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the marker gene.

Using the C2093, B5860Ns or C6055s gene and/or proteins encoded by the genes or transcriptional regulatory region of the genes, compounds can be screened that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene. Such compounds are used as pharmaceuticals for treating or preventing bladder cancer.

Therefore, the present invention provides a method of screening for a compound for treating or preventing bladder cancer using the polypeptide of the present invention. An embodiment of this screening method comprises the steps of: (a) contacting a test compound with a polypeptide encoded by C2093, B5860Ns or C6055s, or an equivalent thereof; (b) detecting the binding activity between the polypeptide and the test compound; and (c) selecting the compound that binds to the polypeptide. In the present invention the polypeptide encoded by C2093, B5860Ns or C6055s, or equivalent thereof may be selected from the group consisting of:

(1) a polypeptide comprising the amino acid sequence of selected from the group consisting of SEQ ID NOs: 2, 4, 6, 130, 132, 134 and 136;

(2) a polypeptide that comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 130, 132, 134 and 136 or a sequence having at least about 80% homology to said sequence; and

(3) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 129, 131, 133 and 135, wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 130, 132, 134 and 136;

The polypeptide of the present invention to be used for screening may be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof. The polypeptide of the present invention to be contacted with a test compound can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.

As a method of screening for proteins, for example, that bind to the polypeptide of the present invention using the polypeptide encoded by C2093, B5860Ns or C6055s of the present invention, many methods well known by a person skilled in the art can be used. Such a screening can be conducted by, for example, immunoprecipitation method, specifically, in the following manner. The C2093, B5860Ns or C6055s gene encoding the polypeptide of the present invention is expressed in host (e.g., animal) cells and so on by inserting the gene to an expression vector for foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8. The promoter to be used for the expression may be any promoter that can be used commonly and include, for example, the SV40 early promoter (Rigby in Williamson (ed.), (1982) Genetic Engineering, vol. 3. Academic Press, London, 83-141), the EF-α promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter (Niwa et al., (1991) Gene 108: 193-9), the RSV LTR promoter (Cullen, (1987) Methods in Enzymology 152: 684-704) the SRα promoter (Takebe et al., (1988) Mol Cell Biol 8: 466-72), the CMV immediate early promoter (Seed and Aruffo, (1987) Proc Natl Acad Sci USA 84: 3365-9), the SV40 late promoter (Gheysen and Fiers, (1982) J Mol Appl Genet 1: 385-94), the Adenovirus late promoter (Kaufman et al., (1989) Mol Cell Biol 9: 946-58), the HSV TK promoter and so on. The introduction of the gene into host cells to express a foreign gene can be performed according to any methods, for example, the electroporation method (Chu et al., (1987) Nucleic Acids Res 15: 1311-26), the calcium phosphate method (Chen and Okayama, (1987) Mol Cell Biol 7: 2745-52), the DEAE dextran method (Lopata et al., (1984) Nucleic Acids Res 12: 5707-17; Sussman and Milman, (1984) Mol Cell Biol 4: 1641-3), the Lipofectin method (Derijard B, et al., (1994) Cell 76: 1025-37; Lamb et al., (1993) Nature Genetics 5: 22-30: Rabindran et al., (1993) Science 259: 230-4) and so on. The polypeptide to be used for screening of the present invention can be expressed as a fusion protein comprising a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C-terminus of the polypeptide of the present invention. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors which can express a fusion protein with, for example, β-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available.

A fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the polypeptide to be used for screening of the present invention by the fusion is also reported. Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the polypeptide to be used for screening of the present invention (Experimental Medicine 13: 85-90 (1995)).

In immunoprecipitation, an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent. The immune complex consists of the polypeptide to be used for screening of the present invention, a polypeptide comprising the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the polypeptide to be used for screening of the present invention, besides using antibodies against the above epitopes, which antibodies can be prepared as described above.

An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody. If the polypeptide to be used for screening of the present invention is prepared as a fusion protein with an epitope, such as GST, an immune complex can be formed in the same manner as in the use of the antibody against the polypeptide to be used for screening of the present invention, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B.

Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, (1988) Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to the polypeptide to be used for screening of the present invention is difficult to detect by a common staining method, such as Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35S-methionine or 35S-cystein, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.

As a method for screening for proteins that bind to a polypeptide of the present invention using the polypeptide, for example, West-Western blotting analysis (Skolnik et al., (1991) Cell 65: 83-90) can be used. Specifically, a protein binding to the polypeptide to be used for screening of the present invention can be obtained by preparing a cDNA library from cells, tissues, organs (for example, tissues such as testis), or cultured cells (e.g., HT1197, UMUC3, J82, HT1376, SW780, RT4 PC3, DU145, or HT1376) expected to express a protein binding to the polypeptide of the present invention using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled polypeptide of the present invention with the above filter, and detecting the plaques expressing proteins bound to the polypeptide of the present invention according to the label. The polypeptide to be used for screening of the invention may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the polypeptide to be used for screening of the present invention, or a peptide or polypeptide (for example, GST) that is fused to the polypeptide of the present invention. Methods using radioisotope or fluorescence and such may be also used.

Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system utilizing cells may be used (“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, (1992) Cell 68: 597-612”, “Fields and Sternglanz, (1994) Trends Genet 10: 286-92”).

In the two-hybrid system, the polypeptide to be used for screening of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express a protein binding to the polypeptide to be used for screening of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide to be used for screening of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.

A compound binding to the polypeptide to be used for screening of the invention can also be screened using affinity chromatography. For example, the polypeptide to be used for screening of the invention may be immobilized on a carrier of an affinity column, and a test compound, containing a protein capable of binding to the polypeptide to be used for screening of the invention, is applied to the column. A test compound herein may be, for example, cell extracts, cell lysates, etc. After loading the test compound, the column is washed, and compounds bound to the polypeptide to be used for screening of the invention can be prepared.

When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.

A biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound compound in the present invention. When such a biosensor is used, the interaction between the polypeptide to be used for screening of the invention and a test compound can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide to be used for screening of the invention and a test compound using a biosensor such as BIAcore.

The methods of screening for molecules that bind when the immobilized polypeptide to be used for screening of the invention is exposed to synthetic chemical compounds, or natural substance banks or a random phage peptide display library, and the methods of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., (1996) Science 273: 458-64; Verdine, (1996) Nature 384: 11-13; Hogan, (1996) Nature 384: 17-9) to isolate not only proteins but chemical compounds that bind to the protein to be used for screening of the invention (including agonist and antagonist) are well known to one skilled in the art.

Alternatively, the present invention provides a method of screening for a compound for treating or preventing bladder cancer using the polypeptide of the present invention encoded by C2093, B5860Ns or C6055s, or an equivalent thereof, comprising the steps as follows:

(a) contacting a test compound with the polypeptide or equivalent thereof;

(b) detecting the biological activity of the polypeptide or equivalent thereof of step (a); and

(c) selecting a compound that suppresses the biological activity of the polypeptide or equivalent thereof in comparison with the biological activity detected in the absence of the test compound.

Since the C2093, B5860Ns and C6055s proteins of the present invention have the activity of promoting cell proliferation of bladder cancer cells, a compound which inhibits this activity can be screened using this activity as an index.

Any polypeptides can be used for screening, so long as they comprise the biological activity of the C2093, B5860Ns or C6055s protein. Such biological activities include the cell-proliferating activity of the human C2093, B5860Ns or C6055s protein. For example, a human C2093, B5860Ns or C6055s protein can be used and polypeptides functionally equivalent to these proteins can also be used. Such polypeptides may be expressed endogenously or exogenously by cells.

The compound isolated by this screening is a candidate for agonists or antagonists of the C2093, B5860Ns or C6055s polypeptide of the present invention. The term “agonist” refers to molecules that activate the function of the polypeptide of the present invention by binding thereto. Likewise, the term “antagonist” refers to molecules that inhibit the function of the polypeptide of the present invention by binding thereto. Moreover, a compound isolated by this screening as “antagonist” is a candidate for compounds which inhibit the in vivo interaction of the polypeptide to be used for screening of the present invention with molecules (including DNAs and proteins).

When the biological activity to be detected in the present method is cell proliferation, it can be detected, for example, by preparing cells which express the polypeptide to be used for screening of the present invention, culturing the cells in the presence of a test compound, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring the colony forming activity as described in the Examples.

In a further embodiment, the present invention provides methods for screening compounds for treating or preventing bladder cancer. As discussed in detail above, by controlling the expression levels of the C2093, B5860Ns and/or C6055s genes, one can control the onset and progression of bladder cancer. Thus, compounds that may be used in the treatment or prevention of bladder cancer can be identified through screenings that use the expression levels of C2093, B5860Ns or C6055s as indices. In the context of the present invention, such screening may comprise, for example, the following steps:

a) contacting a test compound with a cell expressing one or more of the C2093, B5860Ns or C6055s gene; and

b) selecting a compound that reduces the expression level of one or more of the C2093, B5860Ns or C6055s gene in comparison with the expression level detected in the absence of the test compound.

Cells expressing at least one of the one or more of the C2093, B5860Ns or C6055s gene include, for example, cell lines established from bladder cancers; such cells can be used for the above screening of the present invention (e.g., HT1197, UMUC3, J82, HT1376, SW780, RT4 and HT1376). The expression level can be estimated by methods well known to one skilled in the art. In the method of screening, a compound that reduces the expression level of the C2093, B5860N or C6055 genes can be selected as candidate agents to be used for the treatment or prevention of bladder cancer.

Alternatively, the screening method of the present invention may comprise the following steps:

-   -   a) contacting a test compound with a cell into which a vector         comprising the transcriptional regulatory region of one or more         marker genes and a reporter gene that is expressed under the         control of the transcriptional regulatory region has been         introduced, wherein the one or more marker genes are C2093,         B5860Ns or C6055s,     -   b) measuring the expression level or activity of said reporter         gene; and     -   c) selecting a compound that reduces the expression level or         activity of said reporter gene as compared to the expression         level or activity detected in the absence of the test compound.

Suitable reporter genes and host cells are well known in the art. The reporter construct required for the screening can be prepared by using the transcriptional regulatory region of a marker gene. When the transcriptional regulatory region of a marker gene has been known to those skilled in the art, a reporter construct can be prepared by using the previous sequence information. When the transcriptional regulatory region of a marker gene remains unidentified, a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the marker gene.

Examples of supports that may be used for binding proteins include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercial available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column.

The binding of a protein to a support may be conducted according to routine methods, such as chemical bonding and physical adsorption. Alternatively, a protein may be bound to a support via antibodies specifically recognizing the protein. Moreover, binding of a protein to a support can be also conducted by means of avidin and biotin. The binding between proteins is carried out in buffer, for example, but are not limited to, phosphate buffer and Tris buffer, as long as the buffer does not inhibit the binding between the proteins.

In the present invention, a biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound protein. When such a biosensor is used, the interaction between the proteins can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia).

Alternatively, a C2093, B5860N or C6055 polypeptides may be labeled, and the label of the bound protein may be used to detect or measure the bound protein. Specifically, after pre-labeling one of the proteins, the labeled protein is contacted with the other protein in the presence of a test compound, and then bound proteins are detected or measured according to the label after washing.

Labeling substances such as radioisotope (e.g., ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I, ¹³¹I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase), fluorescent substances (e.g., fluorescein isothiosyanete (FITC), rhodamine) and biotin/avidin, may be used for the labeling of a protein in the present method. When the protein is labeled with radioisotope, the detection or measurement can be carried out by liquid scintillation. Alternatively, proteins labeled with enzymes can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.

In case of using an antibody in the present screening, the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance. Alternatively, the antibody against the C2093, B5860Ns or C6055s polypeptide may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance. Furthermore, the antibody bound to the protein in the screening of the present invention may be detected or measured using protein G or protein A column.

Any test compound, including but not limited to, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, syntheticmicromolecular compounds and natural compounds, can be used in the screening methods of the present invention. The test compound of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the “one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection. The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem. 37: 1233-51). Libraries of compounds may be presented in solution (see Houghten (1992) Bio/Techniques 13: 412-21) or on beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9) or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; US Pat. Application 2002103360).

A compound isolated by the screening serves as a candidate for the development of drugs that inhibit the expression of the marker gene or the activity of the protein encoded by the marker gene and can be applied to the treatment or prevention of bladder cancer.

Moreover, compounds in which a part of the structure of the compound inhibiting the activity of proteins encoded by marker genes is converted by addition, deletion and/or replacement are also included as the compounds obtainable by the screening method of the present invention.

When administrating a compound isolated by the method of the present invention as a pharmaceutical for humans and other mammals, including, but not limited to, mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, the isolated compound can be directly administered or can be formulated into a dosage form using known pharmaceutical preparation methods. Pharmaceutical compositions and preparations contemplated by the present invention, as well as methods of making and using same, are described in detail below.

Assessing the Prognosis of a Subject with Bladder Cancer:

The present invention also provides a method of assessing the prognosis of a subject with BLC, including the step of comparing the expression of one or more BLC-associated genes in a test cell population to the expression of the same BLC-associated genes in a reference cell population derived from patients over a spectrum of disease stages. By comparing the gene expression of one or more BLC-associated genes in the test cell population and the reference cell population(s), or by comparing the pattern of gene expression over time in test cell populations derived from the subject, the prognosis of the subject can be assessed.

For example, an increase in the expression of one or more of up-regulated BLC-associated genes, such as those listed in Table 4, as compared to a normal control or a decrease in the expression of one or more of down-regulated BLC-associated genes, such as those listed in Table 5, as compared to a normal control indicates less favorable prognosis. Conversely, a similarity in the expression of one or more of BLC-associated genes listed in Tables 4-5 as compared to normal control indicates a more favorable prognosis for the subject. Preferably, the prognosis of a subject can be assessed by comparing the expression profile of the one or more genes selected from the group consisting of genes listed in Table 4 and 5.

Kits:

The present invention also includes a BLC-detection reagent, e.g., a nucleic acid that specifically binds to or identifies one or more BLC nucleic acids, such as oligonucleotide sequences which are complementary to a portion of a BLC nucleic acid, or an antibody that bind to one or more proteins encoded by a BLC nucleic acid. The detection reagents may be packaged together in the form of a kit. For example, the detection reagents may be packaged in separate containers, e.g., a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix), a control reagent (positive and/or negative), and/or a detectable label. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay may also be included in the kit. The assay format of the kit may be a Northern hybridization or a sandwich ELISA, both of which are known in the art.

For example, a BLC detection reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one BLC detection site. The measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid. A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a separate strip from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of BLC present in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.

Alternatively, the kit may contain a nucleic acid substrate array comprising one or more nucleic acids. The nucleic acids on the array specifically identify one or more nucleic acid sequences represented by the BLC-associated genes listed in Tables 4-5. The expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the nucleic acids represented by the BLC-associated genes listed in Tables 4-5 may be identified by virtue of the level of binding to an array test strip or chip. The substrate array can be on, e.g., a solid substrate, such as a “chip” described in U.S. Pat. No. 5,744,305, the contents of which are incorporated by reference herein in its entirety.

Arrays and Pluralities:

The present invention also includes a nucleic acid substrate array comprising one or more nucleic acids. The nucleic acids on the array specifically correspond to one or more nucleic acid sequences represented by the BLC-associated genes listed in Tables 4-5. The level of expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the nucleic acids represented by the BLC-associated genes listed in Tables 4-5 may be identified by detecting nucleic acid binding to the array.

The present invention also includes an isolated plurality (i.e., a mixture of two or more nucleic acids) of nucleic acids. The nucleic acids may be in a liquid phase or a solid phase, e.g., immobilized on a solid support such as a nitrocellulose membrane. The plurality includes one or more of the nucleic acids represented by the BLC-associated genes listed in Tables 4-5. In various embodiments, the plurality includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the nucleic acids represented by the BLC-associated genes listed in Tables 4-5.

Methods of Inhibiting Bladder Cancer:

The present invention further provides a method for treating or alleviating a symptom of BLC in a subject by decreasing the expression of one or more of the up-regulated BLC-associated genes listed in Table 4 (or the activity of its gene product) or increasing the expression of one or more of the down-regulated BLC-associated genes listed in Table 5 (or the activity of its gene product). Suitable therapeutic compounds can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing BLC. Such subjects can be identified using standard clinical methods or by detecting an aberrant level of expression of one or more of the BLC-associated genes listed in Tables 4-5 or aberrant activity of its gene product. In the context of the present invention, suitable therapeutic agents include, for example, inhibitors of cell cycle regulation, and cell proliferation.

The therapeutic method of the present invention includes the step of increasing the expression, activity, or both of one or more genes or gene products whose expression is decreased (“down-regulated” or “under-expressed” genes) in a BLC cell relative to normal cells of the same tissue type from which the BLC cells are derived. In these methods, the subject is treated with an effective amount of a compound that increases the amount of one or more of the under-expressed (down-regulated) genes in the subject. Administration can be systemic or local. Suitable therapeutic compounds include a polypeptide product of an under-expressed gene, a biologically active fragment thereof, and a nucleic acid encoding an under-expressed gene and having expression control elements permitting expression in the BLC cells; for example, an agent that increases the level of expression of such a gene endogenous to the BLC cells (i.e., which up-regulates the expression of the under-expressed gene or genes). Administration of such compounds counters the effects of aberrantly under-expressed gene or genes in the subject's bladder cancer cells and improves the clinical condition of the subject.

Alternatively, the therapeutic method of the present invention may include the step of decreasing the expression, activity, or both, of one or more genes or gene products whose expression is aberrantly increased (“up-regulated” or “over-expressed” gene) in bladder cancer cells. Expression may be inhibited in any of several ways known in the art. For example, expression can be inhibited by administering to the subject a nucleic acid that inhibits, or antagonizes the expression of the over-expressed gene or genes, e.g., an antisense oligonucleotide or small interfering RNA which disrupts expression of the over-expressed gene or genes.

In yet another embodiment, the therapeutic method includes the step of decreasing the expression or function of the C2093, B5860Ns or C6055s gene. In these methods, the subject is treated with an effective amount of a compound, which decreases the expression and/or activity of one or more of the over-expressed genes (i.e., the C2093, B5860Ns or C6055s gene) in the subject. Administration can be systemic or local. Therapeutic compounds include compounds that decrease the expression level of such gene endogenously existing in the bladder cancerous cells (i.e., compounds that down-regulate the expression of the over-expressed gene(s)). Administration of such therapeutic compounds counter the effects of aberrantly-over expressed gene(s) in the subject's cells and are expected to improve the clinical condition of the subject. Such compounds can be obtained by the screening method of the present invention described above.

The expression of the C2093, B5860Ns or C6055s gene may be also inhibited in any of several ways known in the art including administering to the subject a nucleic acid that inhibits or antagonizes the expression of the gene(s). Antisense oligonucleotides, siRNA or ribozymes which disrupts expression of the gene(s) can be used for inhibiting the expression of the genes.

As noted above, antisense-oligonucleotides corresponding to the nucleotide sequence of the C2093, B5860Ns or C6055s gene can be used to reduce the expression level of the C2093, B5860Ns or C6055s gene. Specifically, the antisense-oligonucleotides of the present invention may act by binding to any of the polypeptides encoded by the C2093, B5860Ns or C6055s gene, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the genes, promoting the degradation of the mRNAs, and/or inhibiting the expression of proteins encoded by the genes, and finally inhibiting the function of the C2093, B5860Ns or C6055s proteins.

An antisense-oligonucleotides and derivatives thereof can be made into an external preparation, such as a liniment or a poultice, by mixing with a suitable base material which is inactive against the derivative and used in the method for treating or preventing bladder cancer of the present invention.

The nucleic acids that inhibit one or more gene products of over-expressed genes also include small interfering RNAs (siRNA) comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid of the nucleotide sequence encoding the C2093, B5860Ns or C6055s gene. Standard techniques of introducing siRNA into the cell can be used in the treatment or prevention of the present invention, including those in which DNA is a template from which RNA is transcribed. The siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin.

Antisense Polynucleotides, Small Interfering RNAs and Ribozymes

As noted above, antisense nucleic acids corresponding to the nucleotide sequence of the BLC-associated genes listed in Table 4 can be used to reduce the expression level of the genes. Antisense nucleic acids corresponding to the BLC-associated genes listed in Table 4 that are up-regulated in bladder cancer are useful for the treatment of bladder cancer. Specifically, the antisense nucleic acids of the present invention may act by binding to the BLC-associated genes listed in Table 4, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the genes, promoting the degradation of the mRNAs, and/or inhibiting the expression of proteins encoded by the BLC-associated genes listed in Table 4, thereby, inhibiting the function of the proteins.

The present invention includes an antisense oligonucleotide that hybridizes with any site within the nucleotide sequence of SEQ ID NO: 3. Specifically, the present invention provides an antisense polynucleotide that hybridizes with nucleic acid comprising the nucleotide sequence from 988 to 1842 of SEQ ID NO: 3, i.e., the region that is specific to the B5860NV1 sequence. This antisense oligonucleotide is preferably against at least about 15 continuous nucleotides of the nucleotide sequence of SEQ ID NO: 3. The above-mentioned antisense oligonucleotide, which contains an initiation codon in the above-mentioned at least 15 continuous nucleotides, is even more preferred.

Derivatives or modified products of antisense oligonucleotides can also be used as antisense oligonucleotides. Examples of such modified products include lower alkyl phosphonate modifications such as methyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioate modifications and phosphoroamidate modifications.

The term “antisense nucleic acids” as used herein encompasses both nucleotides that are entirely complementary to the target sequence and those having a mismatch of one or more nucleotides, so long as the antisense nucleic acids can specifically hybridize to the target sequences. For example, the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least about 70% or higher, preferably at least about 80% or higher, more preferably at least about 90% or higher, even more preferably at least about 95% or higher over a span of at least 15 continuous nucleotides. Algorithms known in the art can be used to determine the homology. Furthermore, derivatives or modified products of the antisense-oligonucleotides can also be used as antisense-oligonucleotides in the present invention. Examples of such modified products include, but are not limited to, lower alkyl phosphonate modifications such as methyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioate modifications and phosphoroamidate modifications.

Such antisense polynucleotides are useful as probes for the isolation or detection of DNA encoding the polypeptide of the invention or as a primer used for amplifications.

The antisense nucleic acids of the present invention act on cells producing the proteins encoded by BLC-associated marker genes by binding to the DNAs or mRNAs encoding the proteins, inhibiting their transcription or translation, promoting the degradation of the mRNAs, and inhibiting the expression of the proteins, thereby resulting in the inhibition of the protein function.

An antisense nucleic acid of the present invention can be made into an external preparation, such as a liniment or a poultice, by admixing it with a suitable base material which is inactive against the nucleic acid.

Also, as needed, the antisense nucleic acids of the present invention can be formulated into, for example, tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such. These can be prepared by following known methods.

The antisense nucleic acids of the present invention can be given to the patient by direct application onto the ailing site or by injection into a blood vessel so that it will reach the site of ailment. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin or derivatives of these.

The dosage of the antisense nucleic acid derivative of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.

The antisense nucleic acids of the present invention inhibit the expression of a protein of the present invention and are thereby useful for suppressing the biological activity of the protein of the invention. In addition, expression-inhibitors, comprising antisense nucleic acids of the present invention, are useful in that they can inhibit the biological activity of a protein of the present invention.

The method of the present invention can be used to alter the expression in a cell of an up-regulated BLC-associated gene, e.g., up-regulation resulting from the malignant transformation of the cells. Binding of the siRNA to a transcript corresponding to one of the BLC-associated genes listed in Table 4 in the target cell results in a reduction in the protein production by the cell. The length of the oligonucleotide is at least 10 nucleotides and may be as long as the naturally-occurring transcript. Preferably, the oligonucleotide is 75, 50, or 25 nucleotides or less in length. Most preferably, the oligonucleotide is about 19 to 25 nucleotides in length.

The antisense nucleic acids of present invention include modified oligonucleotides. For example, thioated oligonucleotides may be used to confer nuclease resistance to an oligonucleotide.

Also, an siRNA against a marker gene can be used to reduce the expression level of the marker gene. Herein, term “siRNA” refers to a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques for introducing siRNA into the cell may be used, including those in which DNA is a template from which RNA is transcribed. In the context of the present invention, the siRNA comprises a sense nucleic acid sequence and an anti-sense nucleic acid sequence against an up-regulated marker gene, such as a BLC-associated gene listed in Table 4. The siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin.

An siRNA of a BLC-associated gene, such as listed in Table 4, hybridizes to target mRNA and thereby decreases or inhibits production of the polypeptides encoded by the BLC-associated gene listed in Table 4 by associating with the normally single-stranded mRNA transcript, thereby interfering with translation and thus, expression of the protein. Thus, siRNA molecules of the invention can be defined by their ability to hybridize specifically to mRNA or cDNA listed in Table 4 under stringent conditions. For the purposes of this invention the terms “hybridize” or “hybridize specifically” are used interchangeably to refer the ability of two nucleic acid molecules to hybridize under “stringent hybridization conditions.” The phrase “stringent hybridization conditions” is discussed above and refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (T_(m)) for the specific sequence at a defined ionic strength pH. The T_(m) is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_(m), 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 50° C.

In the context of the present invention, an siRNA is preferably 500, 200, 100, 50, or 25 nucleotides or less in length. More preferably an siRNA is about 19 to about 25 nucleotides in length. In order to enhance the inhibition activity of the siRNA, nucleotide “u” can be added to 3′ end of the antisense strand of the target sequence. The number of “u”s to be added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5. The added “u”s form single strand at the 3′ end of the antisense strand of the siRNA.

An siRNA of a BLC-associated gene, such as listed in Table 4, can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. In these embodiments, the siRNA molecules of the invention are typically modified as described above for antisense molecules. Other modifications are also possible, for example, cholesterol-conjugated siRNAs have shown improved pharmacological properties. Song et al. Nature Med. 9:347-51 (2003): Alternatively, a DNA encoding the siRNA may be carried in a vector.

Vectors may be produced, for example, by cloning a BLC-associated gene target sequence into an expression vector having operatively-linked regulatory sequences flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee, N. S., et al., (2002) Nature Biotechnology 20:500-5.). An RNA molecule that is antisense strand for mRNA of a BLC-associated gene is transcribed by a first promoter (e.g., a promoter sequence 3′ of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of a BLC-associated gene is transcribed by a second promoter (e.g., a promoter sequence 5′ of the cloned DNA). The sense and antisense strands hybridize in vivo to generate siRNA constructs for silencing of the BLC-associated gene. Alternatively, the two constructs can be utilized to create the sense and antisense strands of an siRNA construct. Cloned BLC-associated genes can encode a construct having secondary structure, e.g., hairpins, wherein a single transcript has both the sense and complementary antisense sequences from the target gene.

A loop sequence, consisting of an arbitrary nucleotide sequence, can be located between the sense and antisense sequence in order to form the hairpin loop structure. Thus, the present invention also provides siRNA having the general formula 5′-[A]-[B]-[A′]-3′, wherein [A] is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes to an mRNA or a cDNA listed in Table 4. In preferred embodiments, [A] is a ribonucleotide sequence corresponding a sequence of gene selected from Table 4,

[B] is a ribonucleotide sequence consisting of about 3 to about 23 nucleotides, and

[A′] is a ribonucleotide sequence consisting of the complementary sequence of [A]. The region [A] hybridizes to [A′], and then a loop consisting of region [B] is formed. The loop sequence may be preferably 3 to 23 nucleotide in length. The loop sequence, for example, can be selected from group consisting of following sequences (http://www.ambion.com/techlib/tb/tb_(—)506.html). Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J.-M., et al., (2002) Nature 418: 435-8.).

CCC, CCACC or CCACACC: Jacque, J. M, et al., (2002) Nature, 418: 435-8.

UUCG: Lee, N. S., et al., (2002) Nature Biotechnology 20:500-5. Fruscoloni, P., et al., (2003) Proc. Natl. Acad. Sci. USA 100(4): 1639-44.

UUCAAGAGA: Dykxhoorn, D. M., et al., (2002) Nature Reviews Molecular Cell Biology 4: 457-67.

For example, preferable siRNAs having hairpin structure of the present invention are shown below. In the following structure, the loop sequence can be selected from group consisting of, CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. Preferable loop sequence is UUCAAGAGA (“ttcaagaga” in DNA).

The nucleotide sequence of suitable siRNAs can be designed using an siRNA design computer program available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). The computer program selects nucleotide sequences for siRNA synthesis based on the following protocol.

Selection of siRNA Target Sites:

-   -   1. Beginning with the AUG start codon of the object transcript,         scan downstream for AA dinucleotide sequences. Record the         occurrence of each AA and the 3′ adjacent 19 nucleotides as         potential siRNA target sites. Tuschl, et al. (1999) Genes Dev 13         (24): 3191-7, don't recommend against designing siRNA to the 5′         and 3′ untranslated regions (UTRs) and regions near the start         codon (within 75 bases) as these may be richer in regulatory         protein binding sites. UTR-binding proteins and/or translation         initiation complexes may interfere with binding of the siRNA         endonuclease complex.     -   2. Compare the potential target sites to the human genome         database and eliminate from consideration any target sequences         with significant homology to other coding sequences. The         homology search can be performed using BLAST, which can be found         on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/.     -   3. Select qualifying target sequences for synthesis. At Ambion,         preferably several target sequences can be selected along the         length of the gene to evaluate.

The regulatory sequences flanking the BLC-associated gene sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. siRNAs are transcribed intracellularly by cloning the BLC-associated gene templates, respectively, into a vector containing, e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter. For introducing the vector into the cell, transfection-enhancing agent can be used. FuGENE (Rochediagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent.

Oligonucleotides and oligonucleotides complementary to various portions of C2093, B5860Ns, or C6055s mRNA were tested in vitro for their ability to decrease production of C2093, B5860Ns, or C6055s in tumor cells (e.g., using the HT1197, UMUC3, J82, HT1376, SW780, RT4 or HT1376 bladder cancer cell line) according to standard methods. A reduction in product of C2093, B5860Ns, or C6055s transcript in cells contacted with the candidate siRNA composition compared to cells cultured in the absence of the candidate composition is detected using C2093, B5860Ns, or C6055s-specific antibodies or other detection strategies. Sequences which decrease production of C2093, B5860Ns, or C6055s in in vitro cell-based or cell-free assays are then tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in in vitro cell-based assay are test in in vivo in rats or mice to confirm decreased C2093, B5860Ns, or C6055s production and decreased tumor cell growth in animals with malignant neoplasms.

Also included in the invention are double-stranded molecules that include the nucleic acid sequence of target sequences, for example, nucleotides 2543-2561 (SEQ ID NO: 21) of SEQ ID NO: 1, nucleotides 2491-2509 of SEQ ID NO: 3 or nucleotides 1639-1657 of SEQ ID NO: 5 (SEQ ID NO: 25), or nucleotides 1905-1923 of SEQ ID NO: 129, nucleotides 1873-1891 of SEQ ID NO: 131, nucleotides 1921-1939 of SEQ ID NO: 133 or nucleotides 2001-2019 of SEQ ID NO: 135 (SEQ ID NO: 144). In the present invention, the double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to SEQ ID NO: 21, 25 or 144, and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand, wherein said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said double-stranded molecule, when introduced into a cell expressing the C2093, B5860Ns, or C6055s gene, inhibits expression of said gene. In the present invention, when the isolated nucleic acid is RNA or derivatives thereof, base “t” should be replaced with “u” in the nucleotide sequences. As used herein, the term “complementary” refers to Watson Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two nucleic acids or compounds or associated nucleic acids or compounds or combinations thereof.

Complementary nucleic acid sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. Furthermore, the sense strand and antisense strand of the isolated nucleotide of the present invention, can form double stranded nucleotide or hairpin loop structure by the hybridization. In a preferred embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In an especially preferred embodiment, where the strands of the duplex are fully complementary, such duplexes contain no mismatches. The nucleic acid molecule is less than 6319 nucleotides (for SEQ ID NO: 1), 5318 nucleotides (for SEQ ID NO: 3), 3851 nucleotides (for SEQ ID NO: 129), 3819 nucleotides (for SEQ ID NO: 131), 3851 nucleotides (for SEQ ID NO: 133) or 3819 nucleotides (for SEQ ID NO: 135) in length. For example, the nucleic acid molecule is 500, 200, or 75 nucleotides or less in length. Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors. The isolated nucleic acids of the present invention are useful for siRNA against C2093, B5860Ns, or C6055s or DNA encoding the siRNA. When the nucleic acids are used for siRNA or coding DNA thereof, the sense strand is preferably longer than about 19 nucleotides, and more preferably longer than about 21 nucleotides.

The antisense oligonucleotide or siRNA of the present invention inhibits the expression of a polypeptide of the present invention and is thereby useful for suppressing the biological activity of a polypeptide of the invention. Also, expression-inhibitors, comprising the antisense oligonucleotide or siRNA of the invention, are useful in the point that they can inhibit the biological activity of the polypeptide of the invention.

Therefore, a composition comprising an antisense oligonucleotide or siRNA of the present invention is useful for treating a bladder cancer. Furthermore, in order to enhance the inhibition activity of the siRNA, nucleotide “u” can be added to 3′ end of the antisense strand of the target sequence. The number of “u”s to be added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5. The added “u”s form single strand at the 3′ end of the antisense strand of the siRNA.

Also, expression-inhibitors, comprising the antisense oligonucleotide or siRNA of the invention, are useful in the point that they can inhibit the biological activity of the polypeptide of the invention. Therefore, a composition comprising the antisense oligonucleotide or siRNA of the present invention is useful in treating a cell proliferative disease such as bladder cancer.

Furthermore, the present invention provides ribozymes that inhibit the expression of the C2093, B5860Ns, or C6055s polypeptide of the present invention.

Generally, ribozymes are classified into large ribozymes and small ribozymes. A large ribozyme is known as an enzyme that cleaves the phosphate ester bond of nucleic acids. After the reaction with the large ribozyme, the reacted site consists of a 5′-phosphate and 3′-hydroxyl group. The large ribozyme is further classified into (1) group I intron RNA catalyzing transesterification at the 5′-splice site by guanosine; (2) group II intron RNA catalyzing self-splicing through a two step reaction via lariat structure; and (3) RNA component of the ribonuclease P that cleaves the tRNA precursor at the 5′ site through hydrolysis. On the other hand, small ribozymes have a smaller size (about 40 bp) compared to the large ribozymes and cleave RNAs to generate a 5′-hydroxyl group and a 2′-3′ cyclic phosphate. Hammerhead type ribozymes (Koizumi et al., (1988) FEBS Lett 228: 228-30) and hairpin type ribozymes (Buzayan, (1986) Nature 323: 349-53; Kikuchi and Sasaki, (1991) Nucleic Acids Res 19: 6751-5) are included in the small ribozymes. Methods for designing and constructing ribozymes are known in the art (see Koizumi et al., (1988) FEBS Lett 228: 228-30; Koizumi et al., (1989) Nucleic Acids Res 17: 7059-71; Kikuchi and Sasaki, (1991) Nucleic Acids Res 19: 6751-5). Thus, ribozymes inhibiting the expression of the polypeptides of the present invention can also be constructed based on their sequence information (SEQ ID NO:1, 3, 5, 129, 131, 133 or 135) and these conventional methods.

Ribozymes against the C2093, B5860Ns, or C6055s transcript inhibit the expression of the over-expressed C2093, B5860Ns, or C6055s protein and can suppress the biological activity of the protein. Therefore, the ribozymes are useful in treating or preventing bladder cancer.

Antibodies:

Alternatively, function of one or more gene products of the genes over-expressed in BLC can be inhibited by administering a compound that binds to or otherwise inhibits the function of the gene products. For example, the compound is an antibody which binds to the over-expressed gene product or gene products.

The present invention refers to the use of antibodies, particularly antibodies against a protein encoded by an up-regulated marker gene, or a fragment of such an antibody. As used herein, the term “antibody” refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto.

The present invention provides an antibody that binds to the polypeptide of the invention. Specifically, the present invention provides an antibody which binds to antigenic determinant comprising the amino acid sequence from 304 to 588 of SEQ ID NO:4, which is the B5860NV1 specific sequence. The antibody of the invention can be used in any form, such as monoclonal or polyclonal antibodies, and includes antiserum obtained by immunizing an animal such as a rabbit with the polypeptide of the invention, all classes of polyclonal and monoclonal antibodies, human antibodies and humanized antibodies produced by genetic recombination.

A polypeptide of the invention used as an antigen to obtain an antibody may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, or rat, more preferably from a human. A human-derived polypeptide may be obtained from the nucleotide or amino acid sequences disclosed herein.

According to the present invention, the polypeptide to be used as an immunization antigen may be a complete protein or a partial peptide of the protein. A partial peptide may comprise, for example, the partial amino acid sequence selected from the B5860NV1 specific sequence (positions from 304 to 588 of SEQ ID NO:4).

Herein, an antibody is defined as a protein that reacts with either the full length or a fragment of a polypeptide of the present invention.

A gene encoding a polypeptide of the invention or its fragment may be inserted into a known expression vector, which is then used to transform a host cell as described herein. The desired polypeptide or its fragment may be recovered from the outside or inside of host cells by any standard method, and may subsequently be used as an antigen. Alternatively, whole cells expressing the polypeptide or their lysates or a chemically synthesized polypeptide may be used as the antigen.

Any mammalian animal may be immunized with the antigen, but preferably the compatibility with parental cells used for cell fusion is taken into account. In general, animals of Rodentia, Lagomorpha or Primates are used. Animals of Rodentia include, for example, mouse, rat and hamster. Animals of Lagomorpha include, for example, rabbit. Animals of Primates include, for example, a monkey of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon and chimpanzees.

Methods for immunizing animals with antigens are known in the art. Intraperitoneal injection or subcutaneous injection of antigens is a standard method for immunization of mammals. More specifically, antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc. If desired, the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion and then administered to mammalian animals. Preferably, it is followed by several administrations of antigen mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days. An appropriate carrier may also be used for immunization. After immunization as above, serum is examined by a standard method for an increase in the amount of desired antibodies.

Polyclonal antibodies against the polypeptides of the present invention may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method. Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies may be isolated from the serum. Immunoglobulin G or M can be prepared from a fraction which recognizes only the polypeptide of the present invention using, for example, an affinity column coupled with the polypeptide of the present invention, and further purifying this fraction using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from spleen. Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.

The above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al. (Galfre and Milstein, (1981) Methods Enzymol 73: 3-46).

Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin and thymidine containing medium). The cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma (non-fused cells), to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.

In addition to the above method, in which a non-human animal is immunized with an antigen for preparing hybridoma, human lymphocytes such as those infected by EB virus may be immunized with a polypeptide, polypeptide expressing cells or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the polypeptide can be obtained (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).

The obtained hybridomas are subsequently transplanted into the abdominal cavity of a mouse and the ascites are extracted. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography or an affinity column to which the polypeptide of the present invention is coupled. The antibody of the present invention can be used not only for purification and detection of the polypeptide of the present invention, but also as a candidate for agonists and antagonists of the polypeptide of the present invention. In addition, this antibody can be applied to the antibody treatment for diseases related to the polypeptide of the present invention. When the obtained antibody is to be administered to the human body (antibody treatment), a human antibody or a humanized antibody is preferable for reducing immunogenicity.

For example, transgenic animals having a repertory of human antibody genes may be immunized with an antigen selected from a polypeptide, polypeptide expressing cells or their lysates. Antibody producing cells are then collected from the animals and fused with myeloma cells to obtain hybridoma, from which human antibodies against the polypeptide can be prepared (see WO92-03918, WO94-02602, WO94-25585, WO96-33735 and WO96-34096).

Alternatively, an immune cell, such as an immunized lymphocyte, producing antibodies may be immortalized by an oncogene and used for preparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, (1990) Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD). For example, a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody. The present invention also provides recombinant antibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment of an antibody or modified antibody, so long as it binds to one or more of the polypeptides of the invention. For instance, the antibody fragment may be Fab, F(ab′)2, Fv or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., (1988) Proc Natl Acad Sci USA 85: 5879-83). More specifically, an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector and expressed in an appropriate host cell (see, for example, Co et al., (1994) J Immunol 152: 2968-76; Better and Horwitz, (1989) Methods Enzymol 178: 476-96; Pluckthun and Skerra, (1989) Methods Enzymol 178: 497-515; Lamoyi, (1986) Methods Enzymol 121: 652-63; Rousseaux et al., (1986) Methods Enzymol 121: 663-9; Bird and Walker, (1991) Trends Biotechnol 9: 132-7).

An antibody may be modified by conjugation with a variety of molecules, such as, for example, polyethylene glycol (PEG). The present invention provides for such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. These modification methods are conventional in the field.

Alternatively, an antibody of the present invention may be obtained as a chimeric antibody, between a variable region derived from a nonhuman antibody and the constant region derived from human antibody, or as a humanized antibody, comprising the complementarity determining region (CDR) derived from a nonhuman antibody, the frame work region (FR) and the constant region derived from a human antibody. Such antibodies can be prepared according to known technology. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see e.g., Verhoeyen et al., (1988) Science 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

Fully human antibodies comprising human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example, in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom & Winter, (1992) J. Mol. Biol. 227:381-8, Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in U.S. Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.

Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, (1988) Cold Spring Harbor Laboratory), but are not limited thereto. A protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS and Sepharose F.F. (Pharmacia).

Exemplary chromatography, with the exception of affinity includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., (1996) Cold Spring Harbor Laboratory Press). The chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and/or immunofluorescence may be used to measure the antigen binding activity of the antibody of the invention. In ELISA, the antibody of the present invention is immobilized on a plate, a polypeptide of the invention is applied to the plate, and then a sample containing a desired antibody, such as culture supernatant of antibody producing cells or purified antibodies, is applied. Then, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme, such as alkaline phosphatase, is applied, and the plate is incubated. Next, after washing, an enzyme substrate, such as p-nitrophenyl phosphate, is added to the plate, and the absorbance is measured to evaluate the antigen binding activity of the sample. A fragment of the polypeptide, such as a C-terminal or N-terminal fragment, may be used as the antigen to evaluate the binding activity of the antibody. BIAcore (Pharmacia) may be used to evaluate the activity of the antibody according to the present invention.

The above methods allow for the detection or measurement of a polypeptide of the invention, by exposing the antibody of the invention to a sample assumed to contain the polypeptide of the invention, and detecting or measuring the immune complex formed by the antibody and the polypeptide.

Because the method of detection or measurement of the polypeptide according to the invention can specifically detect or measure a polypeptide, the method may be useful in a variety of experiments in which the polypeptide is used.

Cancer therapies directed at specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-cancer drugs such as trastuzumab (Herceptin) for the treatment of advanced breast cancer, imatinib methylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F and Tortora G. (2001) Clin Cancer Res.; 7 (10):2958-70. Review.; Slamon D J, et al., (2001) N Engl J Med.; 344 (11):783-92.; Rehwald U, et al., (2003) Blood.; 101 (2):420-4.; Fang G, et al., (2000). Blood, 96, 2246-53.). These drugs are clinically effective and better tolerated than traditional anti-cancer agents because they target only transformed cells. Hence, such drugs not only improve survival and quality of life for cancer patients, but also validate the concept of molecularly targeted cancer therapy. Furthermore, targeted drugs can enhance the efficacy of standard chemotherapy when used in combination with it (Gianni L. (2002). Oncology, 63 Suppl 1, 47-56.; Klejman A, et al., (2002). Oncogene, 21, 5868-76.). Therefore, future cancer treatments will probably involve combining conventional drugs with target-specific agents aimed at different characteristics of tumor cells such as angiogenesis and invasiveness.

These modulatory methods can be performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). The methods involve administering a protein, or combination of proteins, or a nucleic acid molecule, or combination of nucleic acid molecules, as therapy to counteract aberrant expression of the differentially expressed genes or aberrant activity of their gene products.

Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) expression levels or biological activities of genes and gene products, respectively, may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene or genes. Therapeutics that antagonize activity can be administered therapeutically or prophylactically.

Accordingly, therapeutics that may be utilized in the context of the present invention include, e.g., (i) a polypeptide of the over-expressed or under-expressed gene or genes, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to the over-expressed gene or gene products; (iii) nucleic acids encoding the over-expressed or under-expressed gene or genes; (iv) antisense nucleic acids or nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the nucleic acids of one or more over-expressed gene or genes); (v) small interfering RNA (siRNA); or (vi) modulators (i.e., inhibitors, agonists and antagonists that alter the interaction between an over-expressed or under-expressed polypeptide and its binding partner). The dysfunctional antisense molecules are utilized to “knockout” endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, (1989) Science 244: 1288-92).

Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) biological activity may be treated with therapeutics that increase (i.e., are agonists to) activity. Therapeutics that up-regulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a polypeptide (or analogs, derivatives, fragments or homologs thereof) or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).

Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Therapeutic methods of the present invention may include the step of contacting a cell with an agent that modulates one or more of the activities of the gene products of the differentially expressed genes. Examples of agents that modulate protein activity include, but are not limited to, nucleic acids, proteins, naturally-occurring cognate ligands of such proteins, peptides, peptidomimetics, and other small molecule. For example, a suitable agent may stimulate one or more protein activities of one or more differentially under-expressed genes.

Vaccinating Against Bladder Cancer:

The present invention also relates to a method of treating or preventing bladder cancer in a subject comprising the step of administering to said subject a vaccine comprising a polypeptide encoded by a nucleic acid selected from the group consisting of the BLC-associated genes listed in Table 4 (i.e., up-regulated genes), an immunologically active fragment of said polypeptide, or a polynucleotide encoding such a polypeptide or fragment thereof. Administration of the polypeptide induces an anti-tumor immunity in a subject. To induce anti-tumor immunity, a polypeptide encoded by a nucleic acid selected from the group consisting of the BLC-associated genes listed in Table 4, an immunologically active fragment of said polypeptide, or a polynucleotide encoding such a polypeptide or fragment thereof is administered to subject in need thereof. The polypeptide or the immunologically active fragment thereof are useful as vaccines against BLC. In some cases, the proteins or fragments thereof may be administered in a form bound to the T cell receptor (TCR) or presented by an antigen presenting cell (APC), such as macrophage, dendritic cell (DC), or B-cells. Due to the strong antigen presenting ability of DC, the use of DC is most preferable among the APCs.

In the present invention, a vaccine against BLC refers to a substance that has the ability to induce anti-tumor immunity upon inoculation into animals. According to the present invention, polypeptides encoded by the BLC-associated genes listed in Table 4, or fragments thereof, were suggested to be HLA-A24 or HLA-A*0201 restricted epitopes peptides that may induce potent and specific immune response against BLC cells expressing the BLC-associated genes listed in Table 4. Thus, the present invention also encompasses a method of inducing anti-tumor immunity using the polypeptides. In general, anti-tumor immunity includes immune responses such as follows:

-   -   induction of cytotoxic lymphocytes against tumors,     -   induction of antibodies that recognize tumors, and     -   induction of anti-tumor cytokine production.

Therefore, when a certain protein induces any one of these immune responses upon inoculation into an animal, the protein is determined to have anti-tumor immunity inducing effect. The induction of the anti-tumor immunity by a protein can be detected by observing in vivo or in vitro the response of the immune system in the host against the protein.

For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. Specifically, a foreign substance that enters the living body is presented to T cells and B cells by the action of antigen presenting cells (APCs). T cells that respond to the antigen presented by the APCs in an antigen specific manner differentiate into cytotoxic T cells (or cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen, and then proliferate (this is referred to as activation of T cells). Therefore, CTL induction by a certain peptide can be evaluated by presenting the peptide to a T cell via an APC, and detecting the induction of CTLs. Furthermore, APCs have the effect of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells. Since CD4+ T cells and CD8+ T cells are also important in anti-tumor immunity, the anti-tumor immunity-inducing action of the peptide can be evaluated using the activation effect of these cells as indicators.

A method for evaluating the inducing action of CTLs using dendritic cells (DCs) as the APC is well known in the art. DCs are a representative APCs having the strongest CTL-inducing action among APCs. In this method, the test polypeptide is initially contacted with DCs, and then the DCs are contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the test polypeptide has an activity of inducing the cytotoxic T cells. Activity of CTLs against tumors can be detected, for example, using the lysis of ⁵¹Cr-labeled tumor cells as the indicator. Alternatively, the method of evaluating the degree of tumor cell damage using ³H-thymidine uptake activity or LDH (lactose dehydrogenase)-release as the indicator is also well known.

Apart from DCs, peripheral blood mononuclear cells (PBMCs) may also be used as the APC. The induction of CTLs has been reported to be enhanced by culturing PBMCs in the presence of GM-CSF and IL-4. Similarly, CTLs have been shown to be induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

Test polypeptides confirmed to possess CTL-inducing activity by these methods are deemed to be polypeptides having DC activation effect and subsequent CTL-inducing activity. Therefore, polypeptides that induce CTLs against tumor cells are useful as vaccines against tumors. Furthermore, APCs that have acquired the ability to induce CTLs against tumors through contact with the polypeptides are also useful as vaccines against tumors. Furthermore, CTLs that have acquired cytotoxicity due to presentation of the polypeptide antigens by APCs can be also used as vaccines against tumors. Such therapeutic methods for tumors, using anti-tumor immunity due to APCs and CTLs, are referred to as cellular immunotherapy.

Generally, when using a polypeptide for cellular immunotherapy, efficiency of the CTL-induction is known to be increased by combining a plurality of polypeptides having different structures and contacting them with DCs. Therefore, when stimulating DCs with protein fragments, it is advantageous to use a mixture of multiple types of fragments.

Alternatively, the induction of anti-tumor immunity by a polypeptide can be confirmed by observing the induction of antibody production against tumors. For example, when antibodies against a polypeptide are induced in a laboratory animal immunized with the polypeptide, and when growth of tumor cells is suppressed by those antibodies, the polypeptide is deemed to have the ability to induce anti-tumor immunity.

Anti-tumor immunity is induced by administering the vaccine of this invention, and the induction of anti-tumor immunity enables treatment and prevention of BLC. Therapy against cancer or prevention of the onset of cancer includes any of the following steps, such as inhibition of the growth of cancerous cells, involution of cancer, and suppression of the occurrence of cancer. A decrease in mortality and morbidity of individuals having cancer, decrease in the levels of tumor markers in the blood, alleviation of detectable symptoms accompanying cancer, and such are also included in the therapy or prevention of cancer. Such therapeutic and preventive effects are preferably statistically significant. For example, in observation, at a significance level of 5% or less, wherein the therapeutic or preventive effect of a vaccine against cell proliferative diseases is compared to a control without vaccine administration. For example, Student's t-test, the Mann-Whitney U-test, or ANOVA may be used for statistical analysis.

The above-mentioned proteins having immunological activity or a vector encoding such a protein may be combined with an adjuvant. An adjuvant refers to a compound that enhances the immune response against the protein when administered together (or successively) with the protein having immunological activity. Exemplary adjuvants include, but are not limited to, cholera toxin, salmonella toxin, alum, and such, but are not limited thereto. Furthermore, the vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers include, but are not limited to, sterilized water, physiological saline, phosphate buffer, culture fluid, and such. Furthermore, the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants, and such. The vaccine can be administered systemically or locally. Vaccine administration can be performed by single administration, or boosted by multiple administrations.

When using an APC or CTL as the vaccine of this invention, tumors can be treated or prevented, for example, by the ex vivo method. More specifically, PBMCs of the subject receiving treatment or prevention are collected, the cells are contacted with the polypeptide ex vivo, and following the induction of APCs or CTLs, the cells may be administered to the subject. APCs can be also induced by introducing a vector encoding the polypeptide into PBMCs ex vivo. APCs or CTLs induced in vitro can be cloned prior to administration. By cloning and growing cells having high activity of damaging target cells, cellular immunotherapy can be performed more effectively. Furthermore, APCs and CTLs isolated in this manner may be used for cellular immunotherapy not only against individuals from whom the cells are derived, but also against similar types of tumors from other individuals.

Furthermore, a pharmaceutical composition for treating or preventing a cell proliferative disease, such as cancer, comprising a pharmaceutically effective amount of a polypeptide of the present invention is provided. The pharmaceutical composition may be used for raising anti-tumor immunity.

The normal expression of C2093, B5860Ns or C6055s is restricted to testis. Therefore, suppression of this gene may not adversely affect other organs. Thus, the C2093, B5860Ns or C6055s polypeptides are preferable for treating cell proliferative disease, especially bladder cancers. Furthermore, since peptide fragments of proteins specifically expressed in cancerous cells were revealed to induce immune response against the cancer, peptide fragments of C2093, B5860Ns or C6055s can also be used in a pharmaceutical composition for treating or preventing cell proliferative diseases such as bladder cancers. In the present invention, the polypeptide or fragment thereof is administered at a dosage sufficient to induce anti-tumor immunity, which is in the range of 0.1 mg to 10 mg, preferably 0.3 mg to 5 mg, more preferably 0.8 mg to 1.5 mg. The administrations are repeated. For example, 1 mg of the peptide or fragment thereof may be administered 4 times in every two weeks for inducing the anti-tumor immunity.

In addition, polynucleotides encoding C2093, B5860Ns or C6055s, or fragments thereof may be used for raising anti tumor immunity. Such polynucleotides may be incorporated in an expression vector to express C2093, B5860Ns or C6055s, or fragments thereof in a subject to be treated. Thus, the present invention encompasses method for inducing anti tumor immunity wherein the polynucleotides encoding C2093, B5860Ns or C6055s, or fragments thereof are administered to a subject suffering or being at risk of developing cell proliferative diseases such as bladder cancer.

Pharmaceutical Compositions for Inhibiting BLC or Malignant BLC:

The present invention provides compositions for treating or preventing bladder cancer comprising any of the compounds selected by the screening methods of the present invention.

When administrating a compound isolated by the screening methods of the present invention as a pharmaceutical for humans or other mammals, including, but not limited to, mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, chimpanzees, for treating a cell proliferative disease (e.g., bladder cancer) the isolated compound can be directly administered or can be formulated into a dosage form using known pharmaceutical preparation methods. For example, according to the need, the drugs can be taken orally, as sugar-coated tablets, capsules, elixirs and microcapsules; or non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the compounds can be mixed with pharmacologically acceptable carriers or medium, specifically, sterilized water, physiological saline, plant-oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders and such, in a unit dose form required for generally accepted drug implementation. The amount of active ingredients in these preparations makes a suitable dosage within the indicated range acquirable.

Examples of additives that can be mixed to tablets and capsules include, but are not limited to, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry. When the unit dosage form is a capsule, a liquid carrier, such as oil, can also be further included in the above ingredients. Sterile composites for injections can be formulated following normal drug implementations using vehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids including adjuvants, such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, can be used as aqueous solutions for injections. These can be used in conjunction with suitable solubilizers, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants, such as Polysorbate 80™ and HCO-50. Sesame oil or soy-bean oil are examples of oleaginous liquids that may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizers and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol, phenol; and an anti-oxidant. The prepared injection may be filled into a suitable ampoule.

Methods well known to one skilled in the art may be used to administer the inventive pharmaceutical compound to patients, for example as intraarterial, intravenous, percutaneous injections and also as intranasal, intramuscular or oral administrations. The dosage and method of administration vary according to the body-weight and age of a patient and the administration method; however, one skilled in the art can routinely select them. If said compound is encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector administered to perform the therapy. The dosage and method of administration vary according to the body-weight, age, and symptoms of a patient; however, the selection and optimization of these parameters is within the purview of one skilled in the art.

In the context of the present invention, suitable pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration, or for administration by inhalation or insufflation. Preferably, administration is intravenous. The formulations are optionally packaged in discrete dosage units.

Pharmaceutical formulations suitable for oral administration include capsules, cachets or tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, granules, solutions, suspensions and emulsions. The active ingredient is optionally administered as a bolus electuary or paste. Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, fillers, lubricants, disintegrant and/or wetting agents. A tablet may be made by compression or molding, optionally with one or more formulational ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active and/or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), and/or preservatives. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient therein. A package of tablets may contain one tablet to be taken on each of the month.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, optionally contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; as well as aqueous and non-aqueous sterile suspensions including suspending agents and/or thickening agents. The formulations may be presented in unit dose or multi-dose containers, for example as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations suitable for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations suitable for topical administration in the mouth, for example, buccally or sublingually, include lozenges, containing the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles, comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. For intra-nasal administration, the compounds of the invention may be used as a liquid spray, a dispersible powder, or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents and/or suspending agents.

For administration by inhalation the compounds can be conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the compounds may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base, such as lactose or starch. The powder composition may be presented in unit dosage form, for example, as capsules, cartridges, gelatin or blister packs, from which the powder may be administered with the aid of an inhalator or insufflators.

Other formulations include implantable devices and adhesive patches which release a therapeutic agent.

When desired, the above described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions may also contain other active ingredients, such as antimicrobial agents, immunosuppressants and/or preservatives.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art with regard to the type of formulation in question. For example, formulations suitable for oral administration may include flavoring agents.

For example, although there are some differences according to the symptoms, the dose of a compound that binds with the polypeptide of the present invention and regulates its activity is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult (weight 60 kg).

When administering parenterally, in the form of an injection to a normal adult (weight 60 kg), although there are some differences according to the patient, target organ, symptoms and method of administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. Also, in the case of other animals too, it is possible to administer an amount converted to 60 kg of body-weight.

Preferred unit dosage formulations contain an effective dose, as recited below, or an appropriate fraction thereof, of the active ingredient.

For each of the aforementioned conditions, the compositions, e.g., polypeptides and organic compounds, can be administered orally or via injection at a dose ranging from about 0.1 to about 250 mg/kg per day. The dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day. Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, more typically from about 100 mg to about 500 mg.

The dose employed will depend upon a number of factors, including the age and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity. In any event, appropriate and optimum dosages may be routinely calculated by those skilled in the art, taking into consideration the above-mentioned factors.

Furthermore, the present invention provides pharmaceutical compositions for treating or preventing bladder cancer comprising active ingredients that inhibits the expression of the C2093, B5860Ns or C6055s gene. Such active ingredients include antisense polynucleotides, siRNAs or ribozymes against the C2093, B5860Ns or C6055s gene or derivatives, such as expression vector, of the antisense polynucleotides, siRNAs or ribozymes.

The nucleotide sequence of siRNAs may also be designed in the same manner as mentioned above. Furthermore, oligonucleotides and oligonucleotides complementary to various portions of the C2093, B5860Ns or C6055s mRNA may also be selected in the same manner as mentioned above. Examples of C2093, B5860Ns or C6055s siRNA oligonucleotides which inhibit the expression in mammalian cells include the target sequence containing SEQ ID NO: 21, 25 and 144, respectively. The target sequence of SEQ ID NO: 25 is shared between the two B5860N transcripts, B5860NV1 and B5860NV2. Thus, siRNA comprising SEQ ID NO:25 as sense strand may inhibit the expression of both the B5860NV1 and B5860NV2 transcripts. The target sequence of SEQ ID NO: 144 is shared between the four C6055 transcripts, MGC34032, Genbank Accession NO. AK128063, C6055V1 and 6055V2. Thus, siRNA comprising SEQ ID NO:144 as sense strand may inhibit the expression of all the MGC34032, Genbank Accession NO. AK128063, C6055V1 and 6055V2 transcripts. In the present invention, when the nucleic sequence is RNA or derivatives thereof, base “t” should be replaced with “u” in the nucleotide sequences.

The siRNA is directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. Alternatively, the DNA encoding the siRNA is in a vector in the same manner as in the use of the siRNA against the C2093, B5860N or C6055. Furthermore, a loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure. Thus, the present invention also provides siRNA having the general formula 5′-[A]-[B]-[A′]-3′. As mentioned above, in this formula, wherein

[A] is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes to an mRNA or a cDNA of C2093, B5860N or C6055,

[B] is a ribonucleotide sequence consisting of about 3 to about 23 nucleotides, and

[A′] is a ribonucleotide sequence consisting of the complementary sequence of [A].

In the present invention, the siRNA, nucleotide “u” can be added to the 3′ end of [A′], in order to enhance the inhibiting activity of the siRNA. The number of “u”s to be added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5. Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J.-M., et. al., (2002) Nature 418: 435-438.). For example, preferable siRNAs having hairpin structure of the present invention are shown below. In the following structure, the loop sequence can be selected from the group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. Preferable loop sequence is UUCAAGAGA (“ttcaagaga” in DNA) Exemplary hairpin siRNA suitable for use in the context of the present invention include:

for C2093-siRNA (for target sequence of GTGAAGAAGTGCGACCGAA/SEQ ID NO: 21) 5′-gugaagaagugcgaccgaa-[B]-uucggucgcacuucuucac-3′ (SEQ ID NO: 24),

for B5860N-siRNA (for target sequence of CCAAAGTTCCGTAGTCTAA/SEQ ID NO: 25) 5′-ccaaaguuccguagucuaa-[B]-uuagacuacggaacuuugg-3′ (SEQ ID NO: 28) and

for C6055-siRNA (for target sequence of GTTGCAGTTACAGATGAAG/SEQ ID NO: 144) 5′-gttgcagttacagatgaag-[B]-cttcatctgtaactgcaac-3′ (SEQ ID NO: 147)

These active ingredients can be made into an external preparation, such as a liniment or a poultice, by mixing with a suitable base material which is inactive against the derivatives. Also, as needed, they can be formulated into, for example, tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers and such. These can be prepared according to conventional methods.

The active ingredient is given to the patient by directly applying onto the ailing site or by injecting into a blood vessel so that it will reach the site of ailment. A mounting medium can also be used to increase durability and membrane-permeability. Examples of mounting medium includes liposome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives of these.

The dosage of such compositions of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered. Another embodiment of the present invention is a composition for treating or preventing bladder cancer comprising an antibody against a polypeptide encoded by the C2093, B5860Ns or C6055s gene or fragments of the antibody that bind to the polypeptide.

Although there are some differences according to the symptoms, the dose of an antibody or fragments thereof for treating or preventing bladder cancer is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult (weight 60 kg).

When administering parenterally, in the form of an injection to a normal adult (weight 60 kg), although there are some differences according to the condition of the patient, symptoms of the disease and method of administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. Also in the case of other animals too, it is possible to administer an amount converted to 60 kg of body-weight.

Aspects of the present invention are described in the following examples, which are not intended to limit the scope of the invention described in the claims. The following examples illustrate the identification and characterization of genes differentially expressed in BLC cells. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Any patents, patent applications and publications cited herein are incorporated by reference.

EXAMPLES Materials and Methods

Patients, Cell Line, Tissue Samples and Neoadjuvant Chemotherapy.

Human-bladder cancer cell lines HT1197, UMUC3, J82, HT1376, SW780 and RT4 were obtained from ATCC. All cells were cultured in appropriate media; i.e. EMEM (Sigma, St. Louis, Mo.) with 0.1 mM essential amino acid (Roche), 1 mM sodium pyruvate (Roche), 0.01 mg/ml Insulin (Sigma) for HT1197, UMUC3, J82 and HT1376; Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, Calif.) for HBL100, COS7; McCoy's 5a (Sigma) for RT-4; L-15 for SW 780. Each medium was supplemented with 10% fetal bovine serum (Cansera) and 1% antibiotic/antimycotic solution (Sigma). SW 780 cells were maintained at 37° C. an atmosphere of humidified air without CO2. Other cell lines were maintained at 37° C. an atmosphere of humidified air with 5% CO2.

Tissue samples from surgically resected bladder cancers and corresponding clinical information were obtained after each patient had provided written informed consent. A total of 33 cancer samples (9 females, 24 males; median age 66.5 in a range of 53-77 years, except one case with unknown years (BC01025)) (Table 1) that had been confirmed histologically as transitional cell carcinoma of the bladder were selected for this study. Clinical stage was judged according to the UICC TNM classification; we enrolled only patients without node metastasis, T2aN0M0 to T3bN0M0, who were expected to undergo radical cystectomy without prior radiation therapy. Participants were required to have no serious abnormality in renal, hepatic, or hematological function, with ECOG performance status (PS) judged to be <2.

TABLE 1 Patients Examined No. ID Sex Age Stage Grade 1 BC01001 M 62 T3b G3 2 BC01004 M 54 T3b G3 3 BC01007 M 75 T2b G2 > G3 4 BC01009 M 66 T3b G2 5 BC01010 M 77 T2b G1 6 BC01011 M 64 T3b G2 > G3 7 BC01012 M 72 T2b G2 > G3 8 BC01013 F 57 T3b G2 9 BC01014 M 64 T3b G2 10 BC01015 M 57 T3b G3 11 BC01016 M 70 T3b G2 12 BC01017 M 65 T2b G2 13 BC01018 M 71 T3b G2 14 BC01019 M 59 T2b G2 15 BC01020 M 72 T3b G2 16 BC01021 F 68 T2b G2 17 BC01022 M 73 T3b G1 > G2 18 BC01023 F 74 T3b G3 19 BC01024 M 66 T3b G3 20 BC01025 M unknown T2b G2 21 BC01026 M 58 T3b G3 22 BC01027 M 72 T2b G2 23 BC01028 M 69 T3a G2 24 BC01029 F 67 T3a G3 25 BC01031 M 74 T3b G2 26 BC01032 F 64 T3b G3 >> G2 27 BC01033 F 68 T3b G2 28 BC02003 F 53 T3a G2 29 BC02014 M 56 T3b G2 30 BC03001 M 53 T3b G3 31 BC04001 F 70 T3b G3 32 BC05001 F 73 T3a G1 = G2 33 BC05002 M 60 T3b G2

Three to five pieces of cancer tissue were taken from each patient at the time of biopsy prior to neoadjuvant chemotherapy. These samples were immediately embedded in TissueTek OCT medium (Sakura, Tokyo, Japan), frozen, and stored at −80° C. The frozen tissues were sliced into 8-μm sections using a cryostat (Sakura, Tokyo, Japan) and then stained with hematoxylin and eosin for histological examination. Bladder-cancer cells were selectively enriched for our experiments using the EZ-cut system with a pulsed ultraviolet narrow beam-focus laser (SL Microtest GmbH, Germany) according to the manufacturer's protocols. All patients were examined by chest X-ray, computed tomography (CT) and magnetic resonance imaging (MRI) of the abdomen and pelvis, and confirmed to have neither lymph node nor distant metastases.

Extraction of RNA and T7-Based RNA Amplification.

Total RNAs were extracted from each population of microdissected cancer cells, as described previously (Kitahara O, et al., (2001) Cancer Res; 61:3544-9). To guarantee the quality of RNAs, total RNA extracted from the residual tissue of each case were electrophoresed on a denaturing agarose gel, and quality was confirmed by the presence of ribosomal RNA bands. Extraction of total RNA and T7-based RNA amplification were performed as described previously (Okabe H, et al., (2001) Cancer Res; 61:2129-37), except that we used RNeasy Micro Kits (QIAGEN, Valencia, Calif., USA). After two rounds of RNA amplification, we obtained 30-100 μg of amplified RNA (aRNA) for each sample. As a control, normal human bladder poly (A)+ RNA (BD Bioscience, Palo Alto, Calif.), was amplified in the same way. RNA amplified by this method accurately reflects the proportions in the original RNA source, as we had confirmed earlier by semi-quantitative RT-PCR experiments (Kitahara O, et al., (2001) Cancer Res; 61:3544-9), where data from the microarrays were consistent with results from RT-PCR regardless of whether total RNAs or aRNAs were used as templates.

cDNA Microarray.

To obtain cDNAs for spotting on the glass slides, we performed RT-PCR for each gene, as described previously (Kitahara O, et al., (2001) Cancer Res; 61:3544-9). The PCR products were spotted on type VII glass slides (GE Healthcare, Amersham Biosciences, Buckinghamshire UK) with a high-density Microarray Spotter Lucidea (GE Healthcare, Amersham Biosciences); 9,216 genes were spotted in duplicate on a single slide. Three different sets of slides (a total of 27,648 gene spots) were prepared, on each of which the same 52 housekeeping genes and two negative control genes were spotted as well. The cDNA probes were prepared from aRNA in the manner described previously (Okabe H, et al., (2001) Cancer Res; 61:2129-37). For hybridization experiments, 9.0 μg of amplified RNAs (aRNAs) from each cancerous tissue and from the control were reversely transcribed in the presence of Cy5-dCTP and Cy3-dCTP (GE Healthcare, Amersham Biosciences) respectively. Hybridization, washing and detection of signals were carried out as described previously (Okabe H, et al., (2001) Cancer Res; 61:2129-37).

Quantification of Signals.

The signal intensities of Cy3 and Cy5 were quantified from the 27,648 spots and analyzed the signals by substituting backgrounds, using ArrayVision software (Imaging Research, Inc., St. Catharines, Ontario, Canada). Subsequently, the fluorescence intensities of Cy5 (tumor) and Cy3 (control) for each target spot were adjusted so that the mean Cy5/Cy3 ratio of the 52 housekeeping genes became one. Because data derived from low signal intensities are less reliable, we determined a cutoff value on each slide as described previously (Ono K, et al., (2000) Cancer Res; 60:5007-11), and excluded genes from further analysis when both Cy3 and Cy5 dyes yielded signal intensities lower than the cutoff (Saito-Hisaminato A, et al., (2002) DNA Res; 9:35-45). For other genes, the previous method that calculated Cy5/Cy3 as a relative expression ratio using the raw data of each sample was modified, because if either Cy3 or Cy5 signal intensity was lower than the cutoff value the Cy5/Cy3 ratio might provide an extremely high or low reading and lead to selection of false-prediction genes. To reduce that bias, if either Cy3 or Cy5 signal intensity was less than the cutoff value, the Cy5/Cy3 ratios were calculated using half of each cut-off value plus the Cy5 and Cy3 signal intensities of each sample.

Identification of Up- or Down-Regulated Genes in Bladder Cancers.

Up- or down-regulated genes common to bladder cancers were identified and analyzed according to the following criteria. Initially, genes were selected whose relative expression ratio was able to calculate of more than 50% cases and whose expression were up- or down-regulated in more than 50% of cases. Moreover, if the relative expression ratio was able to calculate of 30 to 50% cases, the genes were also evaluated that 80% of cases were up- or down-regulated. The relative expression ratio of each gene (Cy5/Cy3 intensity ratio) was classified into one of four categories as follows: (1) up-regulated (expression ratio was more than 5.0); (2) down-regulated (expression ratio less than 0.2); (3) unchanged expression (expression ratio between 0.2 and 5.0); and (4) not expressed (or slight expression but under the cut-off level for detection). These categories were used to detect a set of genes whose changes in expression ratios were common among samples as well as specific to a certain subgroup. To detect candidate genes that were commonly up- or down-regulated in bladder cancer cell, the overall expression patterns of 27,648 genes were screened to select genes with expression ratios of more than 5.0 or less than 0.2. Among the total of 394 genes that appeared to up-regulated in tumor cells, attention was focused on the ones with in-house identification numbers C2093, B5860N and C6055 because their expression ratios were greater than 5.0 in more than 50% of the informative bladder cancer cases, and showed low expression in normal organs including heart, lung liver and kidney through the expression profiles of normal human tissues.

Semi-Quantitative RT-PCR

The 44 up-regulated genes were selected and examined their expression levels by applying the semi-quantitative RT-PCR experiments. A 3-μg aliquot of aRNA from each sample was reverse-transcribed for single-stranded cDNAs using random primer (Roche) and Superscript II (Invitrogen). Each cDNA mixture was diluted for subsequent PCR amplification with the same primer sets that were prepared for the target DNA- or GAPDH-specific reactions. The primer sequences using RT-PCR in FIG. 1 a are listed in Table 2. The PCR primer sequences using RT-PCR in FIGS. 1 b and 1 c are as follows; 5′-TGCTGGTTCAGAACGAACTATG-3′ (SEQ ID NO. 9) and 5′-TCCTCGTGGCTAATGAAAGC-3′ (SEQ ID NO. 10) for C2093, 5′-GCTACAAGTAAAGAGGGGATGG-3′ (SEQ ID NO. 11) and 5′-GGACAGAAAGGTAAGTCAGTGGG-3′ (SEQ ID NO. 12) for the common sequence of B5860N V1 and V2. Expression of GAPDH served as an internal control. PCR reactions were optimized for the number of cycles to ensure product intensity within the linear phase of amplification (Table 2).

TABLE 2  Primer Sequence for RT-PCR in FIG. 1a SEQ SEQ ID ID LMMID Forward Primer No Reverse Primer No PCR B5860N 5′-ATTGTGGGAATGCACAGG  37 5′-GGACAGAAAGGTAA  12 56d25cy TT-3′ GTCAGTGGG-3′ B0811 5′-GATGTACATATGAGGATT  38 5′-GTCAGTGCACATAAT  39 56d25cy TCCCG-3′ TCCAATAGC-3′ C2093 5′-TTCTAGCTCCTCAACCAA  40 5′-CCGGGAAAGTAAACT  41 56d25cy ATCCT-3′ GACTCAC-3′ F6022 5′-TCTCTTGAGGGCTGCTTTG  42 5′-TCATCCACTGAAATA  43 56d25cy T-3′ CCTGGCTT-3′ F7562 5′-TGGCCATATCAGTTCCAA  44 5′-CTTTGGCATAGCAGC  45 56d25cy CA-3′ CTGAACT-3′ F4976 5′-GGAGAATGAGCTGGATCA  46 5′-ATGCTGCAATTCCCA  47 56d25cy GG-3′ AATCTCT-3′ F6193 5′-AACTCATTGTGTGGCTGT  48 5′-CATCACAATCCTGGG  49 56d25cy GC-3′ AATTCAG-3′ F7409 5′-TCCTGAGGGCCATTTACT  50 5′-TGCATCCAGTAGCTA  51 56d25cy CA-3′ TTCAGCAA-3′ C6055 5′-TCCAGTTGGTTACTCAGT  52 5′-CTGTCATGTGCTCAT  53 56d25cy GTTTG-3′ GTGAGTTT-3′ D5491 5′-CGTCGACAATATAAACAG  54 5′-CGAGCACAAGATAAT  55 56d25cy GGACT-3′ TTTTCCC-3′ C5088 5′-GCAAGTCAGTGCCTAGAT  56 5′-AAAAATTGAGTGTGT  57 56d25cy GGATA-3′ CTCGGTG-3′ D7746 5′-TACAGAGAGGATGGGATT  58 5′-CCTAGCAGTTGTTAG  59 56d25cy GTGTT-3′ AGGCAGAA-3′ A0303 5′-GGGCTTTTAATTTGTGAA  60 5′-TGAAATAGTCTGGCC  61 56d25cy CTTCTG-3′ ATTTGAC-3′ C6865 5′-GTCCCAGACAACAGAAGT  62 5′-AATTTCCTCAGAGCT  63 56d25cy TACCA-3′ CACATACG-3′ F0411 5′-TTTATATTGTGCCATGCAG  64 5′-ACCAGGATCACAGAG  65 56d25cy TCC-3′ AGCTTGA-3′ A8295 5′-TCAGAGTGAGGACTCATT  66 5′-CACAGGGCAGGTTTT  67 56d25cy TATCATTT-3′ GATTTAT-3′ F4025 5′-CCCCTTCAGTGAGCCTCA  68 5′-TGAAATTGACCTGGT  69 56d30cy TA-3′ AGAGCCTT-3′ B2879N 5′-TGTGTTTTCTTTTGGCACC  70 5′-TTACTCCTGGCAAGC  71 56d30cy AT-3′ TGTGAG-3′ A0576N 5′-ATATCAGCATCACGGCAC  72 5′-GTATGATGTAGCTGA  73 56d30cy AA-3′ GGTCCGTG-3′ F6507 5′-TGCTGGCTAACTAAAGAA  74 5′-AAATGAGGCCATTCT  75 56d30cy GATGC-3′ GTTGAGA-3′ F1653 5′-TGAGATTCTGGAGAGTGA  76 5′-TCAGATGTTGTAGCA  77 56d30cy ATGC-3′ GGGACTTT-3′ C2210 5′-CATTTCTTTATAGTTGCCT  78 5′-TTTTGGGTCAGCACT  79 56d30cy CCCC-3′ GACAAT-3′ C7757 5′-GTCTTGGAGGAGCAGATT  80 5′-CTACAATTTATTTCCG  81 56d30cy CCA-3′ AGTCCCC-3′ F5981 5′-CCTCAAGGCCATTGATGT  82 5′-ATGGTAACCACATGA  83 56d25cy AAA-3′ CCCACTG-3′ B9838 5′-AGATAAATCATGACAAGG  84 5′-GCCTTTTGCTTCTTCT  85 56d28cy TCCCC-3′ GTCTTCT-3′ F6910 5′-TTGGTGTAGCACCACACT  86 5′-GCATGACTCAGGGAA  87 56d30cy GG-3′ GGGTATT-3′ D7443 5′-AATGGCATGATCTTGTGT  88 5′-AGATCACTGTGGGTC  89 56d25cy GAAG-3′ TTAAGCAA-3′ C5509 5′-TCTACACCACAGAAAGCA  90 5′-TACCTGAGGAAATTC  91 56d25cy AGTCA-3′ CCGTTACT-3′ A8407 5′-ATAGGGATAATGGCCTCC  92 5′-CTCGCACCTAATAAT  93 56d28cy AATTC-3′ CTGGTCTC-3′ B6283 5′-TGTGTCTCATCTGTGAACT  94 5′-TTCGTGTTACGGTAT  95 56d25cy GCTT-3′ ATCCTGCT-3′ D9407 5′-CCCTAAAGAGTGAGTTTT  96 5′-AAAGGTATTTTCCTG  97 56d25cy CCACA-3′ CAGTAGCC-3′ B2426 5′-GGGCCAGTATGTGTAACT  98 5′-TCAGACATCTGCTGA  99 56d25cy GACAT-3′ CTACAGGA-3′ C1898 5′-CAACGAGAGCAAAACTCC 100 5′-ATAGGGTTTTGCAGT 101 56d25cy AATAC-3′ AGGGAGAG-3′ A7343N 5′-CACATGGTGACCACAGTG 102 5′-AGAGGGTGAGGGCTT 103 56d25cy CAT-3′ TCATCT-3′ D8150 5′-CTTGCTATTGTCAGGTTTT 104 5′-CACTGCATTTACTGCT 105 56d25cy GGTG-3′ TTTGGA-3′ C7747 5′-AGGAGAGGGAGAAATCTT 106 5′-CCAGTTGTATGCCAA 107 56d27cy AGCAA-3′ CATACTCA-3′ F7016 5′-CAGGATTCCAAATGTCAG 108 5′-CCTGCCATTGTCTTTC 109 56d25cy TGAG-3′ AGGTTT-3′ A8317N 5′-CCTATCACAGACGGAAAT 110 5′-TAGGGCAGTTTCCTG 111 56d25cy CCC-3′ TGTTCCT-3′ F6225 5′-TGCTCTGTACATGCCTCTG 112 5′-GCACCCAGAAGGACT 113 56d25cy C-3′ TGCTATT-3′ D6311 5′-CTTCAGAGTGGGTTGGAA 114 5′-TAGTGTGTAATGCGA 115 56d30cy AAAT-3′ TCCTGTGA-3′ C6902 5′-CACTGTGGCAAGATTGCT 116 5′-TACATCACAGCCTTG 117 56d25cy CT-3′ TTCTTTCC-3′ F6333 5′-AAGCGGTCCACAGTCCAA 118 5′-TCACATTGGAGGATA 119 56d30cy TA-3′ GCTGGAA-3′ F7636 5′-GAAGTTTCCTGAGGCTCC 120 5′-GCCCACAAGAGAAGG 121 56d25cy AA-3′ TAGAGGA-3′ F2376 5′-TCCTCTGTCGGTAGCTGTC 122 5′-ACCCTTCATGTTTCTA 123 56d25cy A-3′ GGGCTG-3′ GAPDH 5′-CGACCACTTTGTCAAGCT   7 5′-GGTTGAGCACAGGGT   8 56d20cy CA-3′ ACTTTATT-3′ Northern-Blot Analysis

Northern blots were hybridized with [α32P]-dCTP-labeled amplification products of A0576N, C2093, C5509, B5860N, F1653, B9838 and C6055 prepared by RT-PCR, respectively (Table 3). Specific probes for C6055 were prepared by PCR using a primer set as follows; 5′-CCCCAGTTGAGAGTTTGCTC-3′ (SEQ ID NO: 137) and 5′-CTGTCATGTGCTCATGTGAGTTT-3′ (SEQ ID NO: 53) for the microarray probe of C6055, 5′-TGACATCGGGATTCAGACTAA-3′ (SEQ ID NO: 138) and 5′-AAAGATGCTGGTCCTTGTGC-3′ (SEQ ID NO: 139) for the common region among four transcripts of C6055. Total RNAs were extracted from all bladder cancer cell lines and frozen surgical specimens using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. After treatment with DNase I (Nippon Gene, Osaka, Japan), mRNA was isolated with Micro-FastTrack (Invitrogen) following the manufacturer's instructions. A 1-μg aliquot of each mRNA, along with polyA(+) RNAs isolated from normal adult human heart, lung, liver, kidney, brain, pancreas, testis and bladder (Clontech, Palo Alto, Calif.), were separated on 1% denaturing agarose gels and transferred to nylon membranes. Pre-hybridization, hybridization and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at −80° C. for 14 days.

TABLE 3 Primer Sequence for production of Northern probe by RT-PCR SEQ Sequence ID NO. C2093_F2 5′-TGCTGGTTCAGAACGAACTATG-3′   9 C2093_R2 5′-TCCTCGTGGCTAATGAAAGC-3′  10 B5860N_F2 5′-AGGCAGGCAACTTTCATTTG-3′  13 B5860N_RT 5′-GGACAGAAAGGTAAGTCAGTGG  12 G-3′ A0576N_FT 5′-GTCCCTCATGCCATCACAGTAT-3′ 124 A0576N_RT 5′-GTATGATGTAGCTGAGGTCCGT  73 G-3′ F1653_F1 5′-AGCAGAGGCTGAGCAAAGAG-3′ 125 F1653_R1 5′-CCCCAGTTTCTGGAATGCTA-3′ 126 C5509_F1 5′-AGCGGAGTTCATAAGCCAAA-3′ 127 C5509_R1(RT) 5′-TACCTGAGGAAATTCCCGTTAC  91 T-3′ B9838_F2-RT 5′-TCAAGGGACAATGGTGTGAC-3′ 128 B9838_RT 5′-GCCTTTTGCTTCTTCTGTCTTC  85 T-3′ C6055_F1 5′-CCCCAGTTGAGAGTTTGCTC-3′ 137 C6055_R1 5′-CTGTCATGTGCTCATGTGAGTT  53 T-3′ C6055_F2 5′-TGACATCGGGATTCAGACTAA-3′ 138 C6055_R2 5′-AAAGATGCTGGTCCTTGTGC-3′ 139 5′RACE and 3′RACE

The sequence of 5′ end and 3′ end of B5860N and C6055 was determined by performing 5′ rapid amplification of cDNA ends (5′RACE) and 3′ rapid amplification of cDNA ends (3′RACE) using SMART™ RACE cDNA Amplification Kit (Clontech). The cDNA template was synthesized from bladder cancer cell line, SW780 cells, for amplification and the PCR was carried out using B5860N-specific reverse primer (5′-CATTTTCTGATCCCCACCTCCCTTTG-3′ (SEQ ID NO. 14)), C6055-specific reverse primer (C6055_GSP1; 5′-GATCCAAATGCTAGGGATCCTGTGTG-3′ (SEQ ID NO: 140) and C6055_NGSP1; 5′-CCTGTGTGATATCGTATGGCTCGTCCA-3′ (SEQ ID NO: 141)) for 5′RACE and B5860N-specific forward primer (5′-AGAGGGGATGGGGAAGGTGTTGC-3′ (SEQ ID NO. 15)) for 3′RACE and the AP1 primer supplied in the kit.

Construction of Expression Vectors

The entire coding sequence of C2093, B5860Ns and C6055s cDNA was amplified by PCR using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan) with primers as follows;

C2093-forward, 5′-ATAAGAATGCGGCCGCAATGGAATCTAATTTTAATCAAGAGG-3′ (SEQ ID NO. 16) (the underline indicates NotI site) and

C2093-reverse, 5′-ATAAGAATGCGGCCGCTTTGGCTGTTTTTGTTCGA-3′ (SEQ ID NO. 17) (the underline indicates NotI site),

B5860NV1-forward, 5′-ATAAGAATGCGGCCGCTATGGAGAGTCAGGGTGTGC-3′ (SEQ ID NO. 18) (the underline indicates NotI site) and

B5860NV1-reverse, 5′-CCGCTCGAGTCTTAGACTACGGAACTTTGGT-3′ (SEQ ID NO. 19) (the underline indicates XhoI site),

B5860NV2-forward, 5′-GGAATTCATGGAGAGTCAGGGTGTG-3′ (SEQ ID NO. 20) (the underline indicates EcoRI site) and

B5860NV2-reverse, 5′-CCGCTCGAGTCTTAGACTACGGAACTTTGGT-3′ (SEQ ID NO. 19) (the underline indicates XhoI site),

C6055-forward, 5′-AGAATTCATGATCTTCCTACTGTGTATTATTGGC-3′ (SEQ ID NO: 142) (the underline indicates EcoRI site) and

C6055-reverse, 5′-TATCTCGAGCTGCTTCCTAGTTTGTGGATTTTC-3; (SEQ ID NO: 143) (the underline indicates XhoI site). The PCR products were inserted into the EcoRI and XhoI, and NotI sites of pCAGGSnHA expression vectors, respectively. These constructs were confirmed by DNA sequencing. Western Blotting Analysis

COS7 cells were transiently transfected with 1 μg of pCAGGS-C2093-HA, pCAGGS-B5860NV1-HA, pCAGGS-B5860NV2-HA, or pCAGGS-C6055-HA using FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions, respectively. Cell lysates were separated on 10% SDS-polyacrylamide gels (for pCAGGS-C2093-HA, pCAGGS-B5860NV1-HA, pCAGGS-B5860NV2-HA transfected cells) or 7.5% SDS-polyacrylamide gels (for pCAGGS-C6055-HA transfected cells) and transferred to nitrocellulose membranes, then incubated with a mouse anti-HA antibody (Roche) as primary antibody at 1:1000 dilution. After incubation with sheep anti-mouse IgG-HRP as secondary antibody (Amersham Biosciences), signals were visualized with an ECL kit (Amersham Biosciences).

Immunocytochemical Staining to Detect Exogenous C2093, B5860N and C6055 Proteins in Bladder Cancer Cells

To examine the sub-cellular localization of exogenous C2093, B5860NV1 and B5860NV2, or C6055, COS7 cells were seeded at 1×105 cells per well for all three constructs. After 24 hours, we transiently transfected with 1 μg of pCAGGS-C2093-HA, pCAGGS-B5860NV1-HA, pCAGGS-B5860NV2-HA or pCAGGS-C6055-HA into COS7 cells using FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions, respectively. Then, cells were fixed with PBS containing 4% paraformaldehyde for 15 min, and rendered permeable with PBS containing 0.1% Triton X-100 for 2.5 min at 4° C. Subsequently the cells were covered with 3% BSA in PBS for 12 hours at 4° C. to block non-specific hybridization. Next, each construct-transfected COS7 cells were incubated with a mouse anti-HA antibody (Roche) at 1:1000 dilution. After washing with PBS, both transfected-cells were stained by an Alexa488-conjugated anti-mouse secondary antibody (Molecular Probe) at 1:3000 dilution. Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescent images were obtained under a TCS SP2 AOBS microscope (Leica, Tokyo, Japan).

Synchronization and Flow Cytometry Analysis

HeLa cells (1×106) are transfected with 8 μg of pCAGGS-C2093-HA, or pCAGGS-B5860NV1-HA, pCAGGS-B5860NV2-HA using FuGENE 6 (Roche) according to supplier's protocol. Cells are arrested in G1 phase 24 hours after transfection with aphidicolin (1 ng/ml) for further 16 hours. Cell cycle is released by washing three times with fresh medium and cells are collected at indicated time points. To arrest cells at mitotic phase, cells are incubated with Nocodazole (250 ng/ml) 16 hours before harvest.

For FACS analysis, 400 ml aliquot of synchronized adherent and detached cells were combined and fixed with 70% ethanol at 4° C. After washing with PBS (−) twice, cells were incubated for 30 min with 1 ml of PBS containing 1 mg of RNase I at 37° C. Cells were then stained in 1 ml of PBS containing 50 mg of propidium iodide (PI). The percentages of each fraction of cell cycle phases were determined from at least 10000 cells in a flow cytometer (FACScalibur; Becton Dickinson, San Diego, Calif.).

Construction of C2093, B5860N and C6055 Specific-siRNA Expression Vector using psiU6X3.0

A vector-based RNAi system was established using psiU6BX siRNA expression vector according to the previous report (WO2004/076623). siRNA expression vector against C2093 (psiU6BX-C2093), B5860N (psiU6BX-B5860N), C6055 (psiU6BX-C6055) and control plasmids, psiU6BX-EGFP, -SCR were prepared by cloning of double-stranded oligonucleotides into the BbsI site in the psiU6BX vector. Nucleotide sequences of the double-stranded oligonucleotides are shown below.

C2093si#3 for the target sequence of GTGAAGAAGTGCGACCGAA (SEQ ID NO: 21)

Sense (SEQ ID NO: 22): 5′-CACCGTGAAGAAGTGCGACCGAATTCAAGAGATTCGGTCGCACTT CTTCAC-3′ Antisense (SEQ ID NO: 23) 5′-AAAAGTGAAGAAGTGCGACCGAATCTCTTGAATTCGGTCGCACTT CTTCAC-3′

B5860Nsi#3 for the target sequence of CCAAAGTTCCGTAGTCTAA (SEQ ID NO: 25)

Sense (SEQ ID NO: 26): 5′-CACCCCAAAGTTCCGTAGTCTAATTCAAGAGATTAGACTACGGAA CTTTGG-3′ Antisense (SEQ ID NO: 27): 5′-AAAACCAAAGTTCCGTAGTCTAATCTCTTGAATTAGACTACGGAA CTTTGG-3′

C6055si-08 for the target sequence of GTTGCAGTTACAGATGAAG (SEQ ID NO: 144)

Sense (SEQ ID NO: 145): 5′-CACCGTTGCAGTTACAGATGAAGTTCAAGAGACTTCATCTGTAAC TGCAAC-3′ Antisense (SEQ ID NO: 146): 5′-AAAAGTTGCAGTTACAGATGAAGTCTCTTGAACTTCATCTGTAAC TGCAAC-3′

siRNA control (SiEGFP) for the target sequence of GAAGCAGCACGACTTCTTC (SEQ ID NO: 29)

Sense (SEQ ID NO: 30): 5′-CACCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTG CTGCTTC-3′ Antisense (SEQ ID NO: 31): 5′-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTG CTGCTTC-3′

siRNA control (SiSCR) for the target sequence of GCGCGCTTTGTAGGATTCG (SEQ ID NO: 33)

Sense (SEQ ID NO: 34): 5′-CACCGCGCGCTTTGTAGGATTCGTTCAAGAGACGAATCCTACA AAGCGCGC-3′ Antisense (SEQ ID NO: 35): 5′-AAAAGCGCGCTTTGTAGGATTCGTCTCTTGAACGAATCCTACA AAGCGCGC-3′

These siRNA expression vectors express siRNA having hairpin structure consisting of nucleotide sequence of as follows:

C2093 si#3; (SEQ ID NO: 24) GTGAAGAAGTGCGACCGAATTCAAGAGATTCGGTCGCACTTCTTCAC, B5860N si#3: (SEQ ID NO: 28) CCAAAGTTCCGTAGTCTAATTCAAGAGATTAGACTACGGAACTTTGG, C6055 si-08 (SEQ ID NO: 147) GTTGCAGTTACAGATGAAGTTCAAGAGACTTCATCTGTAACTGCAAC EGFP control: (SEQ ID NO: 32) GAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC, and SCR control: (SEQ ID NO: 36) GCGCGCTTTGTAGGATTCGTTCAAGAGACGAATCCTACAAAGCGCGC Gene-Silencing Effect of C2093, B5860N and C6055

Human bladder cancer cells lines, UMUC3 and J82 for C2093 and B5860N, or SW780 for C6055, were plated onto 10-cm dishes (1×106 cells/dish) and transfected with psiU6BX-EGFP and psiU6BX-SCR as negative controls, psiU6BX-C2093, psiU6BX-B5860N or psiU6BX-C6055 using FuGENE6 (Roche) and Lipofectamine2000 (Invitrogen) reagents for C2093 and B5860N, or using Nucleofector (Amaxa) regent for C6055 according to the supplier's recommendations, respectively. Total RNAs were extracted from the cells at 6 days after the transfection of each construct, and then the knockdown effect of siRNAs was confirmed by semi-quantitative RT-PCR using specific primers for common regions of C2093, B5860N and C6055 as above mentioned. The primers for GAPDH and ACTB as internal control is as follows;

GAPDH (SEQ ID NO. 7) 5′-CGACCACTTTGTCAAGCTCA-3′ and (SEQ ID NO. 8) 5′-GGTTGAGCACAGGGTACTTTATT-3′. ACTB (SEQ ID NO: 148) 5′-CATCCACGAAACTACCTTCAACT-3′ (SEQ ID NO: 149) 5′-TCTCCTTAGAGAGAAGTGGGGTG-3′

Moreover, transfectants expressing siRNAs using UMUC3, J82 and SW780 cell lines were grown for 21, 28 and 24 days in selective media containing 0.6, 1.0 and 0.3 mg/ml of neomycin, respectively. After fixation with 4% paraformaldehyde, transfected cells were stained with Giemsa solution to assess colony formation. MTT assays were performed to quantify cell viability. After 21 and 28 days of culture in the neomycin-containing medium, respectively, MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) was added at a concentration of 0.5 mg/ml. Following incubation at 37° C. for 2.5 or 1.5 hours, acid-SDS (0.01N HCl/10% SDS) was added; the suspension was mixed vigorously and then incubated overnight at 37° C. to dissolve the dark blue crystals. Absorbance at 570 nm was measured with a Microplate Reader 550 (BioRad).

Observation of Multi-Nucleated Cells by C2093-siRNA

After UMUC3 cells were transfected with si-EGFP as negative controls, and si-C2093 using FuGENE6 (Roche), they were cultured and their cellular morphology were observed by microscopy on 7 days after transfection. To further confirm suppression of C2093 protein expression, Western blotting was carried out with anti-C2093 antibody according to the standard protocol.

Anti-C2093 and Anti-B5860N Antibodies

Plasmids expressing partial fragments of C2093 (1612-1780 a.a.) (SEQ ID NO: 150) and B5860NV2 (337-527 a.a) or B5860NV1 (621-811 a.a.) (SEQ ID NO: 151) that contained His-tag at their COOH-terminals were prepared using pET21 vector, respectively. The recombinant proteins were expressed in Escherichia coli, BL21 codon-plus strain (Stratagene, La Jolla, Calif.), and purified using Ni-NTA resin and TALON according to the supplier's protocols. The proteins were inoculated into rabbits; the immune sera were purified on affinity columns according to standard protocols. Affinity-purified anti-C2093 and anti-B5860N antibodies were used for Western blotting, immunoprecipitation, and immunostaining

Immunocytochemical Staining to Detect Endogenous C2093 and B5860N Proteins in Bladder Cancer Cells

To examine the subcellular localization of endogenous C2093 or B5860N, we seeded UMUC3 cells that expressed C2093 or B5860N endogenously at 1×105 cells per well, respectively. After 24 hours, cells were fixed with PBS containing 4% paraformaldehyde for 15 min, and rendered permeable with PBS containing 0.1% Triton X-100 for 2 min at 4° C. Subsequently the cells were covered with 3% BSA in PBS for 12 hours at 4° C. to block non-specific hybridization. Next, UMUC3 cells were incubated with affinity-purified anti-C2093 antibody or anti-B5860N antibody at 1:100 dilution. Nuclei were counter-stained with 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI). After washing with PBS, UMUC3 cells were stained by an Alexa488-conjugated anti-rabbit secondary antibody (Molecular Probe) at 1:1000 dilution. Fluorescent images were obtained under a confocal microscope (Leica, Tokyo, Japan).

Immunohistochemical Staining to Detect Endogenous C2093 and B5860N Proteins in Normal or Bladder Cancer Tissues

Slides of paraffin-embedded normal adult human tissues (BioChain, Hayward, Calif.) and surgical bladder cancer specimens were stained using ENVISION+ Kit/HRP (DakoCytomation, Glostrup, Denmark) after the sections were deparaffinized and warmed with the microwave oven for 5 minutes at 80° C. in antigen retrieval solution with high pH (DAKO) using anti-C2093 antibody, or the sections were deparaffinized and autocraved for 15 minutes at 108° C. in antigen retrieval solution with high pH (DAKO) using anti-B5860N antibody, respectively. After blocking of endogenous peroxidase and proteins, these sections were incubated with affinity-purified anti-C2093 or anti-B5860N antibodies at 1:20 dilution. Immunodetection was done with peroxidase-labeled anti-rabbit immunoglobulin (Envision kit, Dako Cytomation, Carpinteria, Calif.). Finally, the reactants were developed with 3,3 V-diaminobenzidine (Dako) and the cells were counterstained with hematoxylin.

Results

To obtain precise expression profiles of bladder cancers, only bladder cancer cells with LMM were collected. The proportion of cancer cells selected by this procedure was estimated to be nearly 100%, as determined by microscopic visualization (data not shown).

394 up-regulated genes whose expression ratio was more than 5.0 were identified (Table 4). Of these genes, the 288 functionally characterized genes that were over-expressed in bladder cancer cells were included, and the other 106 (including 51 ESTs) were currently unknown. These up-regulated elements included significant genes involved in signal transduction pathway, oncogenes, cell cycle, and cell adhesion and cytoskeleton. On the other hand, 1272 down-regulated genes whose expression ratio was less than 0.2 were identified (Table 5). Of these down-regulated genes, the 1026 functionally characterized genes that were down-regulated in bladder cancer cells were included, and the other 246 (including 119 ESTs) were unknown.

To confirm the expression pattern of these up-regulated genes in bladder cancers, semi-quantitative RT-PCR analysis was performed using bladder cancer cell lines and normal human tissues including normal bladder and normal transitional cells. Comparing the ratios of the expression levels of the 44 up-regulated genes whose expression were over-expressed in almost of all informative cases, the results were highly similar to those of the microarray analysis in the great majority of the tested cases (FIG. 1). Particularly, B5860N, B0811, C2093, F6022, F4976, D5491, F0411, D7746, A0576N, F1653, C2210, C7757 and D7443 also showed no expression in normal vital organs. These data verified the reliability of our strategy to identify commonly up-regulated genes in bladder cancer cells.

To further examine the expression pattern of these up-regulated genes, A0576N, C2093, C5509, B5860N, F1653, B9838 and C6055, Northern blot analyses were performed with bladder cancer cell lines using each [α32P]-dCTP-labeled amplification products of A0576N, C2093, C5509, B5860N, F1653, B9838 and C6055 prepared by RT-PCR using each primer set as shown in Table 3 as each probe (FIG. 2). All of them also showed much higher expression in bladder cancer cell lines than that in normal bladder. Particularly, A0576N, C2093, B5860N, B9838 and C6055 were specifically over-expressed in bladder cancer cell lines, but were not expressed in normal human tissues including normal bladder. These suggest that these specifically over-expressed genes might be good candidate for molecular target-therapy.

TABLE 4 Up-regulated gene in bladder cancer BLC assignment LMMID ACCESSION GENE TITLE 1 C3760 BC008718 BIRC5 Baculoviral IAP repeat-containing 5 (survivin) 2 D9621 NM_178229 IQGAP3 IQ motif containing GTPase activating protein 3 3 F1653 BC011621 HOOK1 Hook homolog 1 (Drosophila) 4 C8776 AA766028 AF15Q14 AF15q14 protein 5 F6507 AL046246 PGAP1 GPI deacylase 6 F3374 AF195765 RAMP RA-regulated nuclear matrix- associated protein 7 A3555 K02581 TK1 Thymidine kinase 1, soluble 8 C5005 BX648571 FLJ38736 Hypothetical protein FLJ38736 9 D0006 NM_145697 CDCA1 Cell division cycle associated 1 10 B8706 R52614 CDK5R1 Cyclin-dependent kinase 5, regulatory subunit 1 (p35) 11 A4693 U42408 LAD1 Ladinin 1 12 E0388 BC033193 MGC30208 Hypothetical protein MGC30208 13 B8882 BC005832 KIAA0101 KIAA0101 14 D4920 AI247180 GUCY1B2 Guanylate cyclase 1, soluble, beta 2 15 F2861 CR598555 KIF20A Kinesin family member 20A 16 E1412 AI989840 17 E1349 BC041395 Homo sapiens, Similar to diaphanous homolog 3 (Drosophila), clone IMAGE: 5277415, mRNA 18 A3243 CR624652 TTK TTK protein kinase 19 E1138 AF318349 LEMD2 LEM domain containing 2 20 B0259 AA234962 PKP3 Plakophilin 3 21 A2921 NM_002391 MDK Midkine (neurite growth-promoting factor 2) 22 A3058 NM_202002 FOXM1 Forkhead box M1 23 B5904 BC008947 C10orf3 Chromosome 10 open reading frame 3 24 D6767 BM312795 Transcribed locus 25 A4388 NM_001988 EVPL Envoplakin 26 D4636 AF370395 EPS8L1 EPS8-like 1 27 B4587 AB096683 MGC57827 Similar to RIKEN cDNA 2700049P18 gene 28 A1618 X70683 SOX4 SRY (sex determining region Y)-box 4 29 F6022 AK022479 HDHD1A Haloacid dehalogenase-like hydrolase domain containing 1A 30 A0061 AF053306 BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) 31 E2104 CN280172 CDNA clone IMAGE: 4734740, partial cds 32 A2254 NM_006845 KIF2C Kinesin family member 2C 33 B8870 NM_018685 ANLN Anillin, actin binding protein (scraps homolog, Drosophila) 34 A3896 BC015050 OIP5 Opa-interacting protein 5 35 A8287 R87657 DKFZp762E1312 Hypothetical protein DKFZp762E1312 36 A1835 U18018 ETV4 Ets variant gene 4 (E1A enhancer binding protein, E1AF) 37 D2882 AA777954 38 A2282 BC014039 MELK Maternal embryonic leucine zipper kinase 39 C7457 W44613 LY6K CDNA for differentially expressed CO16 gene 40 E1774 AK022881 KIAA1272 KIAA1272 protein 41 E0785 BC039269 NALP2 NACHT, leucine rich repeat and PYD containing 2 42 F1332 CR592757 BRRN1 Barren homolog (Drosophila) 43 F7562 AI146812 44 C2093 H93085 MPHOSPH1 M-phase phosphoprotein 1 45 F7083 AL044366 RUNX1 Runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene) 46 F6312 AW977404 SLC15A2 Solute carrier family 15 (H+/peptide transporter), member 2 47 E0954 BC021290 IMP-2 IGF-II mRNA-binding protein 2 48 A5657 BQ219156 HSPC150 HSPC150 protein similar to ubiquitin-conjugating enzyme 49 F7407 AF095288 PTTG2 Pituitary tumor-transforming 2 50 B2909 CR625760 TOP2A Topoisomerase (DNA) II alpha 170 kDa 51 F2445 AK022644 MGC3101 Hypothetical protein MGC3101 52 B9303 AK129960 LOC92558 Hypothetical protein LOC92558 53 C6902 BC007608 HMGB3 High-mobility group box 3 54 A0499 BM912233 CKS2 CDC28 protein kinase regulatory subunit 2 55 A9518N AA570186 Hypothetical gene supported by AK096951; BC066547 56 A2728 AK126687 LLGL2 Lethal giant larvae homolog 2 (Drosophila) 57 A5623 AF044588 PRC1 Protein regulator of cytokinesis 1 58 B6283 AY257469 CIT Citron (rho-interacting, serine/threonine kinase 21) 59 C8054 NM_001445 FABP6 Fatty acid binding protein 6, ileal (gastrotropin) 60 E0465 BC010044 CDC20 CDC20 cell division cycle 20 homolog (S. cerevisiae) 61 B0811 AW183154 KIF14 Kinesin family member 14 62 E1010 CN341726 2′-PDE 2′-phosphodiesterase 63 F4976 AF165527 DGCR8 DiGeorge syndrome critical region gene 8 64 F2376 AK021714 CDNA FLJ11652 fis, clone HEMBA1004461 65 C0234 NM_020639 RIPK4 Receptor-interacting serine-threonine kinase 4 66 A5953 N24235 KIAA0789 KIAA0789 gene product 67 B7725 NM_031966 CCNB1 Cyclin B1 68 A0576N NM_138555 KIF23 Kinesin family member 23 69 B7480 AF407165 PPP1R14C Protein phosphatase 1, regulatory (inhibitor) subunit 14C 70 B4218 BQ052480 IFI27 Interferon, alpha-inducible protein 27 71 F1655 AL137343 NSE1 NSE1 72 C9468 AA885242 KIFC2 Kinesin family member C2 73 A1787 NM_016343 CENPF Centromere protein F, 350/400ka (mitosin) 74 D6683 NM_003106 SOX2 SRY (sex determining region Y)-box 2 75 A0024 AF017790 KNTC2 Kinetochore associated 2 76 D9407 CR749484 LOC152519 Hypothetical protein LOC152519 77 F6910 BF940192 KIAA0776 KIAA0776 78 A1367 D14520 KLF5 Kruppel-like factor 5 (intestinal) 79 C2210 AI755171 MRCL3 Myosin regulatory light chain MRCL3 80 C5088 R62589 SEMA3C Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C 81 F1242 AB020713 NUP210 Nucleoporin 210 82 C7747 CA314541 Transcribed locus 83 D5753 AA971042 RHPN1 Rhophilin, Rho GTPase binding protein 1 84 E1801 AW971869 SORL1 Sortilin-related receptor, L(DLR class) A repeats-containing 85 A1063 BU600928 SPRR1B Small proline-rich protein 1B (cornifin) 86 A8317N BQ013695 FLJ10420 Hypothetical protein FLJ10420 87 B5860N BM683578 DEPDC1 DEP domain containing 1 88 F3847 AK027006 TNRC9 Trinucleotide repeat containing 9 89 A0018 NM_198433 STK6 Serine/threonine kinase 6 90 A4356 Y00503 KRT19 Keratin 19 91 A0004 AB003698 CDC7 CDC7 cell division cycle 7 (S. cerevisiae) 92 D7212 AA132702 XTP2 HBxAg transactivated protein 2 93 F4584 XM_049695 VANGL2 Vang-like 2 (van gogh, Drosophila) 94 E1619 BU620217 NS Nucleostemin 95 B6905 BU675191 CGI-72 CGI-72 protein 96 C6486 X83618 HMGCS2 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 2 (mitochondrial) 97 A3298 M91670 UBE2S Ubiquitin-conjugating enzyme E2S 98 C0488 AA781195 PRAME Preferentially expressed antigen in melanoma 99 A2691N BC041846 CDH3 Cadherin 3, type 1, P-cadherin (placental) 100 F0757 AK023834 CDCP1 CUB domain-containing protein 1 101 F6193 AK026280 Transcribed locus, weakly similar to XP_375099.1 hypothetical protein LOC283585 [Homo sapiens] 102 A0303 U79240 PASK PAS domain containing serine/threonine kinase 103 C7757 AK024506 C14orf80 Chromosome 14 open reading frame 80 104 F2228 X51688 CCNA2 Cyclin A2 105 D9210 CA844321 MGC3196 Hypothetical protein MGC3196 106 F6225 AW970636 FOXP1 Forkhead box P1 107 C7353 AK122903 EPS8L2 EPS8-like 2 108 E1776 AI138333 MAP4K3 Mitogen-activated protein kinase kinase kinase kinase 3 109 D5491 AA947258 Transcribed locus 110 E0885 NM_183047 PRKCBP1 Protein kinase C binding protein 1 111 B4479 AF258572 GSDML Gasdermin-like 112 C6719 BC013892 PVRL4 Poliovirus receptor-related 4 113 A2796 NM_006681 NMU Neuromedin U 114 D8458 AA830668 115 F6333 AW590215 SLC4A8 Solute carrier family 4, sodium bicarbonate cotransporter, member 8 116 D4789 AW070371 SIMP Source of immunodominant MHC- associated peptides 117 A4875 NM_000336 SCNN1B Sodium channel, nonvoltage-gated 1, beta (Liddle syndrome) 118 A0143N AJ606319 MYB V-myb myeloblastosis viral oncogene homolog (avian) 119 B2426 CB116740 POGK Pogo transposable element with KRAB domain 120 F7685 AV699624 Transcribed locus 121 C5020 BC004352 KIF22 Kinesin family member 22 122 C7114 BU738386 LOC284352 Hypothetical protein LOC284352 123 F7399 AI928242 TFCP2L1 Transcription factor CP2-like 1 124 A6002 AA179812 FLJ21918 Hypothetical protein FLJ21918 125 A2439 AF053305 BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) 126 A1591 NM_002534 OAS1 2′,5′-oligoadenylate synthetase 1, 40/46 kDa 127 F6689 AK021848 128 C7737 AA969163 Transcribed locus 129 F0411 AW898615 130 B6548 R61700 131 A5136N AF133086 ST14 Suppression of tumorigenicity 14 (colon carcinoma, matriptase, epithin) 132 D6877 AI002358 133 B4478 AA910946 AP1M2 Adaptor-related protein complex 1, mu 2 subunit 134 C9858 NM_006892 DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta 135 B1194 AK090897 GPATC2 G patch domain containing 2 136 A4438 AF055015 EYA2 Eyes absent homolog 2 (Drosophila) 137 A7602 CD359557 DAZAP2 DAZ associated protein 2 138 D7746 AI024928 C13orf24 Chromosome 13 open reading frame 24 139 C1901 AA649063 FLJ21865 Endo-beta-N-acetylglucosaminidase 140 E0639 AK023937 THEA Thioesterase, adipose associated 141 A7322 CA314912 LOC202451 Hypothetical protein LOC202451 142 B2879N AA173525 Similar to Zinc finger protein Rlf (Rearranged L-myc fusion gene protein) (Zn-15 related protein) 143 B7367 CR616479 AMACR Alpha-methylacyl-CoA racemase 144 F4070 NM_020897 HCN3 Hyperpolarization activated cyclic nucleotide-gated potassium channel 3 145 C4658 AF132541 GMIP GEM interacting protein 146 B8739 AA992910 VSIG2 V-set and immunoglobulin domain containing 2 147 F7003 AW291323 148 B2454 AL833191 SMC2L1 SMC2 structural maintenance of chromosomes 2-like 1 (yeast) 149 A1223 X73502 KRT20 Keratin 20 150 F0969 AK026201 RAB3IP RAB3A interacting protein (rabin3) 151 F3726 AF098668 LYPLA2 Lysophospholipase II 152 A1209 NM_001071 TYMS Thymidylate synthetase 153 C3759 NM_003072 SMARCA4 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 154 C0328 CR592555 Full-length cDNA clone CS0DE011YI04 of Placenta of Homo sapiens (human) 155 B6353 R19310 RELN Reelin 156 C1555 AK094156 Similar to KIAA0454 protein 157 D7443 AI017753 AHI1 Abelson helper integration site 158 D8001 AW976634 Transcribed locus 159 F7409 AI739486 MFAP3 Microfibrillar-associated protein 3 160 F6200 AK023542 161 B4626 BC052574 FLJ20171 Hypothetical protein FLJ20171 162 F7636 AI651388 163 A2310 AF261758 DHCR24 24-dehydrocholesterol reductase 164 C9450 AI338875 CDK3 Cyclin-dependent kinase 3 165 F3973 AL157504 MRNA; cDNA DKFZp586O0724 (from clone DKFZp586O0724) 166 C7681 AA151182 ZNF339 Zinc finger protein 339 167 D9500 AI361654 168 C9098 AY376439 ECT2 Epithelial cell transforming sequence 2 oncogene 169 B5152N CR618521 CSNK1E Casein kinase 1, epsilon 170 D4223 AK095136 RASGEF1A RasGEF domain family, member 1A 171 F2073 NM_020990 CKMT1 Creatine kinase, mitochondrial 1 (ubiquitous) 172 B7466 AA128378 KIAA0303 KIAA0303 protein 173 A7083 CR608243 ARHGAP8 Rho GTPase activating protein 8 174 D5363 AI419859 GALNT7 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase 7 (GalNAc-T7) 175 A9044 BC003186 Pfs2 DNA replication complex GINS protein PSF2 176 C8682 NM_005329 HAS3 Hyaluronan synthase 3 177 A1605 NM_203401 STMN1 Stathmin 1/oncoprotein 18 178 A4045N BE538546 PMCH Pro-melanin-concentrating hormone 179 A0042 AF029082 SFN Stratifin 180 A1524 U29344 FASN Fatty acid synthase 181 F3821 AL117612 MAL2 Mal, T-cell differentiation protein 2 182 C4276 NM_001407 CELSR3 Cadherin, EGF LAG seven-pass G- type receptor 3 (flamingo homolog, Drosophila) 183 D7936 BQ022849 TEX27 Testis expressed sequence 27 184 A5159 BC080193 ERBB2 V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) 185 A4542 NM_001305 CLDN4 Claudin 4 186 F8586 AA579871 SMARCC1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 1 187 A7870 NM_018492 TOPK T-LAK cell-originated protein kinase 188 B7330N BM726315 GALNT6 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase 6 (GalNAc-T6) 189 A1054 M13755 G1P2 Interferon, alpha-inducible protein (clone IFI-15K) 190 A6139 BU730831 PAFAH1B3 Platelet-activating factor acetylhydrolase, isoform Ib, gamma subunit 29 kDa 191 A6127 AI356291 GPT2 Glutamic pyruvate transaminase (alanine aminotransferase) 2 192 C8580 CR611223 CLDN7 Claudin 7 193 C6055 AA001450 MGC34032 Hypothetical protein MGC34032 194 F8392 AI432199 LMO4 LIM domain only 4 195 A1970 BC000356 MAD2L1 MAD2 mitotic arrest deficient-like 1 (yeast) 196 D8466 AI619500 Transcribed locus 197 C9305 AI080640 AGR2 Anterior gradient 2 homolog (Xenopus laevis) 198 E0170 BQ417235 Transcribed locus 199 F7497 AW973864 SYNJ2BP Synaptojanin 2 binding protein 200 D7200 BC069011 TRA2A Transformer-2 alpha 201 F4522 AK023400 DCL-1 Type I transmembrane C-type lectin receptor DCL-1 202 B8930 AA513445 RBM21 RNA binding motif protein 21 203 A4643 J05428 UGT2B7 UDP glycosyltransferase 2 family, polypeptide B7 204 F8409 BC041096 CLCA2 Chloride channel, calcium activated, family member 2 205 A2603N Z46629 SOX9 SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal) 206 A6979 AI357616 LOC90133 Hypothetical protein LOC90133 207 E1348 BX640908 EVI1 Ecotropic viral integration site 1 208 F5885 AK023050 209 A3644 NM_006949 STXBP2 Syntaxin binding protein 2 210 C5509 NM_201269 Zep-2 Zinc finger motif enhancer binding protein 2 211 B2579N N70341 ELAC2 ElaC homolog 2 (E. coli) 212 D4039 CB142087 MARVELD2 MARVEL domain containing 2 213 A1800 NM_052987 CDK10 Cyclin-dependent kinase (CDC2-like) 10 214 F3387 AK126185 PPFIA4 Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 4 215 A2735 BC036811 PTHR2 Parathyroid hormone receptor 2 216 C9937 BC013048 C8orf20 Chromosome 8 open reading frame 20 217 F7614 AI114655 LOC284058 Hypothetical protein LOC284058 218 B1561 AK074562 QKI Quaking homolog, KH domain RNA binding (mouse) 219 B4400 BX647949 FRAS1 Fraser syndrome 1 220 C4588 AA016977 MRNA; cDNA DKFZp686F1844 (from clone DKFZp686F1844) 221 D3205 AY024361 MLL3 B melanoma antigen family, member 4 222 B7272 BQ268701 223 B7706 R22536 FLJ13052 NAD kinase 224 G0074 AK127891 MGC10744 Hypothetical protein MGC10744 225 C0909 U38276 SEMA3F Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F 226 A8777 BM990713 IL28RA Interleukin 28 receptor, alpha (interferon, lambda receptor) 227 E1497 BU625507 SLC16A3 Solute carrier family 16 (monocarboxylic acid transporters), member 3 228 C6865 BC041417 Transcribed locus, moderately similar to NP_955751.1 potassium channel regulator [Homo sapiens] 229 A0327N NM_002421 MMP1 Matrix metalloproteinase 1 (interstitial collagenase) 230 C8624 NM_005858 AKAP8 A kinase (PRKA) anchor protein 8 231 B9838 AA018510 MGC33382 Hypothetical protein MGC33382 232 B4649 BM996064 TJP3 Tight junction protein 3 (zona occludens 3) 233 F7985 AA682421 234 B5490 AB014555 HIP1R Huntingtin interacting protein-1- related 235 A0959 NM_001034 RRM2 Ribonucleotide reductase M2 polypeptide 236 A5644 BC015582 MGC23280 Hypothetical protein MGC23280 237 B7163 AA262462 NT5C2 5′-nucleotidase, cytosolic II 238 F5981 AL050119 TMEM1 Transmembrane protein 1 239 F7087 AL043093 FAM47B Family with sequence similarity 47, member B 240 C6634 AA398740 CDNA FLJ41168fis, clone BRACE 2041095 241 C0285 AK093343 FLJ23231 Hypothetical protein FLJ23231 242 C0417 AF311320 SLC37A1 Solute carrier family 37 (glycerol-3- phosphate transporter), member 1 243 F3641 AY099469 SLAC2-B SLAC2-B 244 C1730 BU682808 GNAS GNAS complex locus 245 F2351 AL162042 246 C3640 NM_182641 FALZ Fetal Alzheimer antigen 247 B7032N AA398096 PFKFB4 6-phosphofructo-2-kinase/fructose- 2,6-biphosphatase 4 248 D8837 NM_012189 CABYR Calcium-binding tyrosine-(Y)- phosphorylation regulated (fibrousheathin 2) 249 F6161 BF056203 ABHD7 Abhydrolase domain containing 7 250 B5458N AA889610 CARHSP1 Calcium regulated heat stable protein 1, 24 kDa 251 D3452 BX482647 PARP14 Poly (ADP-ribose) polymerase family, member 14 252 B6562 CA306079 PLEKHJ1 Pleckstrin homology domain containing, family J member 1 253 C6789 AK125177 LOC149134 Hypothetical protein LOC149134 254 C0485 BC064568 LOC150223 Hypothetical protein LOC150223 255 A7343N N68578 LIPC Lipase, hepatic 256 A6349 AK095197 PAQR6 Progestin and adipoQ receptor family member VI 257 A1865 U60808 CDS1 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1 258 A7352 AJ421269 TD-60 RCC1-like 259 F0534 NM_004360 CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 260 F3919 AK025341 FARP1 FERM, RhoGEF (ARHGEF) and pleckstrin domain protein 1 (chondrocyte-derived) 261 F7374 AI656728 ARIH1 Ariadne homolog, ubiquitin- conjugating enzyme E2 binding protein, 1 (Drosophila) 262 A2822 BQ015859 CSTA Cystatin A (stefin A) 263 E1344 BC064421 C2orf29 Chromosome 2 open reading frame 29 264 A1604 X52186 ITGB4 Integrin, beta 4 265 D8789 AI025912 GLCCI1 Glucocorticoid induced transcript 1 266 D3350 R45979 267 F7016 BE179023 FLJ11142 Hypothetical protein FLJ11142 268 A0636 Z29066 NEK2 NIMA (never in mitosis gene a)- related kinase 2 269 A5223 BC007379 MGC16207 Hypothetical protein MGC16207 270 G0445 AK000981 271 B0869N AF274048 UHRF1 Ubiquitin-like, containing PHD and RING finger domains, 1 272 C7864 D84454 SLC35A2 Solute carrier family 35 (UDP- galactose transporter), member A2 273 A2837 BU618918 CDKN3 Cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase) 274 C7435 BC029267 MUC20 Mucin 20 275 A4383 Z97029 RNASEH2A Ribonuclease H2, large subunit 276 F7162 AK000364 CHD7 Chromodomain helicase DNA binding protein 7 277 B1819 AY165122 MYH14 Myosin, heavy polypeptide 14 278 A0207 M73812 CCNE1 Cyclin E1 279 D8150 BF965334 PRKRA Protein kinase, interferon-inducible double stranded RNA dependent activator 280 C8051 BM685415 C10orf116 Chromosome 10 open reading frame 116 281 A5601 H19339 MRNA; cDNA DKFZp547G036 (from clone DKFZp547G036) 282 B7365 BC025755 C6orf134 Chromosome 6 open reading frame 134 283 A1957 U20979 CHAF1A Chromatin assembly factor 1, subunit A (p150) 284 B5103N AI091425 VGLL1 Vestigial like 1 (Drosophila) 285 F2779 BC001226 PLEK2 Pleckstrin 2 286 C6110 W67193 GFPT1 Glutamine-fructose-6-phosphate transaminase 1 287 A4616 AJ007669 FANCG Fanconi anemia, complementation group G 288 A1859N NM_001002295 GATA3 GATA binding protein 3 289 A0333 NM_002466 MYBL2 V-myb myeloblastosis viral oncogene homolog (avian)-like 2 290 A6869 BC011665 TCF3 Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) 291 C7801 AI299827 TFCP2L3 Transcription factor CP2-like 3 292 F8081 BF433219 293 A3587 NM_003088 FSCN1 Fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus) 294 A6935 AA523117 DC-TM4F2 Tetraspanin similar to TM4SF9 295 F3642 CR619487 DKFZP564C103 DKFZP564C103 protein 296 F3549 AK025185 297 F6831 AK024988 Similar to KIAA0160 gene product is novel. 298 E0499 BM906554 COX6B1 Cytochrome c oxidase subunit Vib polypeptide 1 (ubiquitous) 299 A7770 R55185 IRX3 Iroquois homeobox protein 3 300 A2865 AJ297436 PSCA Prostate stem cell antigen 301 B3762 BC035311 ZD52F10 Dermokine 302 F8140 AW976457 MBNL1 Muscleblind-like (Drosophila) 303 A8295 AA430571 Transcribed locus 304 F8687 AW081894 305 A1166 S62028 RCV1 Recoverin 306 B8658 CA429220 SKP2 S-phase kinase-associated protein 2 (p45) 307 B6813 BX092653 Transcribed locus 308 E0909 BU726646 Transcribed locus 309 D1287 BC012136 ISL2 ISL2 transcription factor, LIM/homeodomain, (islet-2) 310 A6151 BU620959 RAPGEFL1 Rap guanine nucleotide exchange factor (GEF)-like 1 311 A6486 W67936 RAI RelA-associated inhibitor 312 G0008 AK026743 C21orf96 Chromosome 21 open reading frame 96 313 F3089 AB046838 KIAA1618 KIAA1618 314 F3293 AL389951 NUP50 Nucleoporin 50 kDa 315 B5870 AI312573 CPNE3 Copine III 316 A9334 BC039343 HN1 Hematological and neurological expressed 1 317 B9951 NM_005556 KRT7 Keratin 7 318 B8627 R39044 RAB27B RAB27B, member RAS oncogene family 319 D2335 BQ018544 Hypothetical LOC389908 320 B4097 CR596974 MLP MARCKS-like protein 321 F7332 AI936859 RTKN Rhotekin 322 B8205 AL133100 FLJ20531 Hypothetical protein FLJ20531 323 C2132 AW134658 MSI2 Musashi homolog 2 (Drosophila) 324 E0491 BC062785 CDNA clone IMAGE: 4734740, partial cds 325 A4959 AF042282 EXO1 Exonuclease 1 326 A1824 NM_002224 ITPR3 Inositol 1,4,5-triphosphate receptor, type 3 327 A1007 Z29093 DDR1 Discoidin domain receptor family, member 1 328 C4330 BC006000 CAPNS2 Calpain, small subunit 2 329 D6311 BI771102 PHYHIPL Family with sequence similarity 13, member C1 330 B5994 T81301 AFURS1 ATPase family homolog up-regulated in senescence cells 331 C6374 AA493372 LOC55971 Insulin receptor tyrosine kinase substrate 332 F7512 AW978905 HNRPK Heterogeneous nuclear ribonucleoprotein K 333 F0864 AK025277 TNRC6 Trinucleotide repeat containing 6 334 A9568 BC022217 C6orf85 Chromosome 6 open reading frame 85 335 F2807 AL080146 CCNB2 Cyclin B2 336 A0587 NM_006739 MCM5 MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. cerevisiae) 337 B1119 AI215478 HMMR Hyaluronan-mediated motility receptor (RHAMM) 338 B7060 BC067795 MGC11308 Hypothetical protein MGC11308 339 B8276 BC009831 RAB25 RAB25, member RAS oncogene family 340 A8043 W72411 TP73L Tumor protein p73-like 341 B9340 T78186 DNMT3A DNA (cytosine-5-)-methyltransferase 3 alpha 342 B4456 BX537652 FLJ12892 Hypothetical protein FLJ12892 343 B3796 AA116022 USP18 Ubiquitin specific protease 18 344 E0950 BF740209 PYGB Phosphorylase, glycogen; brain 345 B4409 XM_371116 MYO5B Myosin VB 346 C1898 AL713801 SLAMF7 SLAM family member 7 347 F4025 AK021428 C6orf210 Chromosome 6 open reading frame 210 348 F0983 AL832106 MLR2 Ligand-dependent corepressor 349 A3256 L07597 RPS6KA1 Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 350 A1715 M74178 MST1 Macrophage stimulating 1 (hepatocyte growth factor-like) 351 A8407 CB988759 C2orf33 Chromosome 2 open reading frame 33 352 A6363 CR621577 Homo sapiens, clone IMAGE: 5301514, mRNA 353 B4618 BM014054 LOC339229 Hypothetical protein LOC339229 354 D9773 BC039118 STX6 Syntaxin 6 355 A0309 U85658 TFAP2C Transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) 356 F4885 NM_003681 PDXK Pyridoxal (pyridoxine, vitamin B6) kinase 357 A0516 BC064662 TRAF2 TNF receptor-associated factor 2 358 B2664 AA682861 PARD6B Par-6 partitioning defective 6 homolog beta (C. elegans) 359 A1874 CR617220 KRT8 Keratin 8 360 A2608 NM_002230 JUP Junction plakoglobin 361 A5157 AF027153 362 A1767 M93107 BDH 3-hydroxybutyrate dehydrogenase (heart, mitochondrial) 363 B4853N CD013889 CHRNA1 Cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) 364 A5044 AK127479 SPINT2 Serine protease inhibitor, Kunitz type, 2 365 A4467 AF038961 MPDU1 Mannose-P-dolichol utilization defect 1 366 B3995 BC073757 KRT18 Keratin 18 367 D5376 BQ946404 CALM2 Calmodulin 2 (phosphorylase kinase, delta) 368 A2620 NM_001649 APXL Apical protein-like (Xenopus laevis) 369 A0437 NM_005782 THOC4 THO complex 4 370 B5141 NM_194463 RNF128 Ring finger protein 128 371 B9661 BF764924 WSB1 WD repeat and SOCS box-containing 1 372 C8847 AA232990 Transcribed locus 373 C0023 NM_002744 PRKCZ Protein kinase C, zeta 374 D6248 AW295407 FLJ25078 Hypothetical protein FLJ25078 375 A2088 BF131641 S100A11 S100 calcium binding protein A11 (calgizzarin) 376 D8834 BM729250 GTF3A General transcription factor IIIA 377 A1139 AF230388 TRIM29 Tripartite motif-containing 29 378 C9024 AI678218 AE2 Hypothetical protein AE2 379 E0571 BG115155 FLJ10726 Hypothetical protein FLJ10726 380 C4878 BC040176 LOC130576 Hypothetical protein LOC130576 381 E0516 AK075185 KDELR1 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 1 382 B6529 CA314443 PLXNA3 Plexin A3 383 A2111 BC062996 DBI Diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein) 384 A6657 BX451670 FLJ30525 Hypothetical protein FLJ30525 385 F5784 AK022067 KIAA1217 KIAA1217 386 A7182 NM_003731 SSNA1 Sjogren's syndrome nuclear autoantigen 1 387 A4144 BC004376 ANXA8 Annexin A8 388 A2382 NM_004456 EZH2 Enhancer of zeste homolog 2 (Drosophila) 389 A9467 BC045658 LOC57228 Hypothetical protein from clone 643 390 F6419 AW978490 SSH2 Slingshot homolog 2 (Drosophila) 391 B3971 AF290612 NUSAP1 Nucleolar and spindle associated protein 1 392 B4325 BC053605 LOC440448 393 A6441 AI279896 CGI-69 CGI-69 protein 394 C7625 BU684240 EHF Ets homologous factor

TABLE 5 Down-regulated gene in bladder cancer BLC assignment LMMID ACCESSION GENE TITLE 395 A0898 BC011393 CHN1 Chimerin (chimaerin) 1 396 A0944 Z24725 PLEKHC1 Pleckstrin homology domain containing, family C (with FERM domain) member 1 397 A1750 D31716 BTEB1 Basic transcription element binding protein 1 398 A1852 M19713 TPM1 Tropomyosin 1 (alpha) 399 A2460 AF000959 CLDN5 Claudin 5 (transmembrane protein deleted in velocardiofacial syndrome) 400 A2701 NM_003028 SHB SHB (Src homology 2 domain containing) adaptor protein B 401 A6184 NM_133268 OSBPL1A Oxysterol binding protein-like 1A 402 A3188 M27110 PLP1 Proteolipid protein 1 (Pelizaeus- Merzbacher disease, spastic paraplegia 2, uncomplicated) 403 A3340 M93284 PNLIPRP2 Pancreatic lipase-related protein 2 404 A4189 AA922716 PRKACB Protein kinase, cAMP-dependent, catalytic, beta 405 A4472 AF042081 SH3BGRL SH3 domain binding glutamic acid- rich protein like 406 A5084 CR614015 CD14 CD14 antigen 407 A5498 BX093242 Transcribed locus 408 A5356 NM_001002260 C9orf58 Chromosome 9 open reading frame 58 409 A5795 BQ775444 CORO1C Coronin, actin binding protein, 1C 410 A5704 AB018254 KIAA0711 KIAA0711 gene product 411 A0100 NM_002006 FGF2 Fibroblast growth factor 2 (basic) 412 A6111 NM_018105 THAP1 THAP domain containing, apoptosis associated protein 1 413 A1365 D10653 TM4SF2 Transmembrane 4 superfamily member 2 414 A1764 NM_002526 NT5E 5′-nucleotidase, ecto (CD73) 415 A2964 BQ219660 GNG11 Guanine nucleotide binding protein (G protein), gamma 11 416 A3203 NM_002436 MPP1 Membrane protein, palmitoylated 1, 55 kDa 417 A3322 M80899 AHNAK AHNAK nucleoprotein (desmoyokin) 418 A3739 NM_000090 COL3A1 Collagen, type III, alpha 1 (Ehlers- Danlos syndrome type IV, autosomal dominant) 419 A3748 X51593 MYH3 Myosin, heavy polypeptide 3, skeletal muscle, embryonic 420 A4630 U89281 RODH 3-hydroxysteroid epimerase 421 A4972 NM_002487 NDN Necdin homolog (mouse) 422 A5807 W80773 CDNA FLJ13601 fis, clone PLACE1010069 423 A5937 BC028315 GABARAPL1 GABA(A) receptor-associated protein like 1 424 A5785 CR627469 PSMB7 Proteasome (prosome, macropain) subunit, beta type, 7 425 A1572 NM_015833 ADARB1 Adenosine deaminase, RNA- specific, B1 (RED1 homolog rat) 426 A1847 U31525 GYG Glycogenin 427 A1879 U45955 GPM6B Glycoprotein M6B 428 A2452 BX537488 CSRP1 Cysteine and glycine-rich protein 1 429 A2978 X04741 UCHL1 Ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) 430 A3946 NM_021738 SVIL Supervillin 431 A4579 L29394 HP Haptoglobin 432 A4473 BX648582 SPRY2 Sprouty homolog 2 (Drosophila) 433 A4611 S79851 TXNRD1 Thioredoxin reductase 1 434 A5118 X17576 NCK1 NCK adaptor protein 1 435 A5888 U56417 AGPAT1 1-acylglycerol-3-phosphate O- acyltransferase 1 (lysophosphatidic acid acyltransferase, alpha) 436 A6099 W60630 JAM3 Junctional adhesion molecule 3 437 A1083 BX510904 MYH2 Myosin, heavy polypeptide 2, skeletal muscle, adult 438 A2610 NM_020546 ADCY2 Adenylate cyclase 2 (brain) 439 A2972 X72475 Amyloid immunoglobulin light chain protein BRE 440 A3214 X17042 PRG1 Proteoglycan 1, secretory granule 441 A5485 NM_018357 FLJ11196 Acheron 442 A0260 U47413 CCNG1 Cyclin G1 443 A0232 NM_006219 PIK3CB Phosphoinositide-3-kinase, catalytic, beta polypeptide 444 A0946 U62961 OXCT1 3-oxoacid CoA transferase 1 445 A1617 NM_133378 TTN Titin 446 A1855 X73114 MYBPC1 Myosin binding protein C, slow type 447 A1891 BC038984 GAS6 Growth arrest-specific 6 448 A2372 AF458589 PPP1R12A Protein phosphatase 1, regulatory (inhibitor) subunit 12A 449 A2739 AF073920 RGS6 Regulator of G-protein signalling 6 450 A3733 X04665 THBS1 Thrombospondin 1 451 A5211 R55332 LRIG1 Leucine-rich repeats and immunoglobulin-like domains 1 452 A5251 NM_025164 KIAA0999 KIAA0999 protein 453 A5694 BM996053 C10orf9 Chromosome 10 open reading frame 9 454 A0094 NM_002293 LAMC1 Laminin, gamma 1 (formerly LAMB2) 455 A0383 M13690 SERPING1 Serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1, (angioedema, hereditary) 456 A0791 X63556 FBN1 Fibrillin 1 (Marfan syndrome) 457 A1064 NM_024164 TPSB2 Tryptase, alpha 458 A0960 U60115 FHL1 Four and a half LIM domains 1 459 A1736 NM_001456 FLNA Filamin A, alpha (actin binding protein 280) 460 A2031 NM_003040 SLC4A2 Solute carrier family 4, anion exchanger, member 2 (erythrocyte membrane protein band 3-like 1) 461 A2388 BC000574 PCOLCE Procollagen C-endopeptidase enhancer 462 A2860 BC002436 STX4A Syntaxin 4A (placental) 463 A4328 NM_000573 CR1 Complement component (3b/4b) receptor 1, including Knops blood group system 464 A4602 X63679 TRAM1 Translocation associated membrane protein 1 465 A4974 NM_006063 KBTBD10 Kelch repeat and BTB (POZ) domain containing 10 466 A5015 NM_001451 FOXF1 Forkhead box F1 467 A0941 S59049 RGS1 Regulator of G-protein signalling 1 468 A1753 BC063289 C4A Complement component 4B 469 A1882 AF018081 COL18A1 Collagen, type XVIII, alpha 1 470 A2595 BC010839 RPN1 Ribophorin I 471 A2224 NM_004469 FIGF C-fos induced growth factor (vascular endothelial growth factor D) 472 A2740 CR607883 CDO1 Cysteine dioxygenase, type I 473 A3089 AK091961 UMOD Uromodulin (uromucoid, Tamm- Horsfall glycoprotein) 474 A4193 BU737730 RBP1 Retinol binding protein 1, cellular 475 A4841 AF037261 SCAM-1 Vinexin beta (SH3-containing adaptor molecule-1) 476 A5514 AA669799 ASMTL Acetylserotonin O- methyltransferase-like 477 A1085 BQ073704 LGALS1 Lectin, galactoside-binding, soluble, 1 (galectin 1) 478 A0837 L02950 CRYM Crystallin, mu 479 A0961 NM_001482 GATM Glycine amidinotransferase (L- arginine:glycine amidinotransferase) 480 A1592 NM_000177 GSN Gelsolin (amyloidosis, Finnish type) 481 A2043 BC005330 TFPI2 Tissue factor pathway inhibitor 2 482 A2272 AF195530 XPNPEP1 X-prolyl aminopeptidase (aminopeptidase P) 1, soluble 483 A6080 N99340 CLIPR-59 CLIP-170-related protein 484 A0357 X15606 ICAM2 Intercellular adhesion molecule 2 485 A0775 L12579 CUTL1 Cut-like 1, CCAAT displacement protein (Drosophila) 486 A1074 D90228 ACAT1 Acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl Coenzyme A thiolase) 487 A1610 NM_002084 GPX3 Glutathione peroxidase 3 (plasma) 488 A1452 CD013947 ITGB6 Integrin, beta 6 489 A1754 AB119995 CES1 Carboxylesterase 1 (monocyte/macrophage serine esterase 1) 490 A3061 U07643 LTF Lactotransferrin 491 A2715 BC035802 GZMK Granzyme K (serine protease, granzyme 3; tryptase II) 492 A2751 M68874 PLA2G4A Phospholipase A2, group IVA (cytosolic, calcium-dependent) 493 A3563 NM_021136 RTN1 Reticulon 1 494 A4201 CR592913 RRAS2 Related RAS viral (r-ras) oncogene homolog 2 495 A4237 BC058074 WISP2 WNT1 inducible signaling pathway protein 2 496 A4709 BC016952 CYR61 Cysteine-rich, angiogenic inducer, 61 497 A4871 Z19002 ZBTB16 Zinc finger and BTB domain containing 16 498 A6057 BC035939 MRAS Muscle RAS oncogene homolog 499 A5773 N72174 EGFL5 EGF-like-domain, multiple 5 500 A0533 NM_003932 ST13 Suppression of tumorigenicity 13 (colon carcinoma) (Hsp70 interacting protein) 501 A1748 U29089 PRELP Proline arginine-rich end leucine- rich repeat protein 502 A2478 Y13647 SCD Stearoyl-CoA desaturase (delta-9- desaturase) 503 A6225 AJ420439 MRNA full length insert cDNA clone EUROIMAGE 1585492 504 A3965 AF078695 REV3L REV3-like, catalytic subunit of DNA polymerase zeta (yeast) 505 A6264 BC022522 CD200 CD200 antigen 506 A4365 U68494 SLC30A1 Solute carrier family 30 (zinc transporter), member 1 507 A4491 L15388 GRK5 G protein-coupled receptor kinase 5 508 A4983 X12830 IL6R Interleukin 6 receptor 509 A4769 AF004563 STXBP1 Syntaxin binding protein 1 510 A4887 NM_001173 ARHGAP5 Rho GTPase activating protein 5 511 A6081 AK023172 C2orf23 Chromosome 2 open reading frame 23 512 A0090 BC040499 TGFBR2 Transforming growth factor, beta receptor II (70/80 kDa) 513 A0905 X14723 CLU Clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone- repressed prostate message 2, apolipoprotein J) 514 A0635 NM_004329 BMPR1A Bone morphogenetic protein receptor, type IA 515 A1474 M86406 ACTN2 Actinin, alpha 2 516 A1453 M37721 PAM Peptidylglycine alpha-amidating monooxygenase 517 A1886 BC029261 MYOC Myocilin, trabecular meshwork inducible glucocorticoid response 518 A1995 M14745 BCL2 B-cell CLL/lymphoma 2 519 A3435 CR623240 PSG9 Pregnancy specific beta-1- glycoprotein 9 520 A3866 AF080157 CHUK Conserved helix-loop-helix ubiquitous kinase 521 A4111 BC033040 SLC1A1 Solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 522 A3834 AB010419 CBFA2T3 Core-binding factor, runt domain, alpha subunit 2; translocated to, 3 523 A3959 AF055081 DES Desmin 524 A4076 BC008837 AKR1B10 Aldo-keto reductase family 1, member B10 (aldose reductase) 525 A3738 NM_002332 LRP1 Low density lipoprotein-related protein 1 (alpha-2-macroglobulin receptor) 526 A4586 D86977 DHX38 DEAH (Asp-Glu-Ala-His) box polypeptide 38 527 A5806 BC042960 Transcribed locus, moderately similar to NP_787073.2 hypothetical protein MGC35023 [Homo sapiens] 528 A0386 K02215 AGT Angiotensinogen (serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 8) 529 A0415 M77349 TGFBI Transforming growth factor, beta- induced, 68 kDa 530 A0922 NM_004394 DAP Death-associated protein 531 A1594 NM_002422 MMP3 Matrix metalloproteinase 3 (stromelysin 1, progelatinase) 532 A2471 NM_001155 ANXA6 Annexin A6 533 A3114 M95585 HLF Hepatic leukemia factor 534 A4742 AF019214 HBP1 HMG-box transcription factor 1 535 A5262 NM_020182 TMEPAI Transmembrane, prostate androgen induced RNA 536 A5911 AK125888 FBXO32 F-box protein 32 537 A6082 N66336 ALS2CR15 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 14 538 A0159 BC028049 PPP3CB Protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform (calcineurin A beta) 539 A0423 NM_006744 RBP4 Retinol binding protein 4, plasma 540 A0192 M62829 EGR1 Early growth response 1 541 A1378 NM_000362 TIMP3 Tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory) 542 A2275 L80005 SNRPN SNRPN upstream reading frame 543 A2508 X03350 ADH1B Alcohol dehydrogenase IB (class I), beta polypeptide 544 A2188 J02770 IF I factor (complement) 545 A2542 J02874 FABP4 Fatty acid binding protein 4, adipocyte 546 A3037 BC030975 IL1RL1 Interleukin 1 receptor-like 1 547 A6208 NM_004264 SURB7 SRB7 suppressor of RNA polymerase B homolog (yeast) 548 A4040 NM_181425 FXN Frataxin 549 A4254 NM_001848 COL6A1 Collagen, type VI, alpha 1 550 A4407 AF055872 TNFSF13 Tumor necrosis factor (ligand) superfamily, member 12 551 A5853 N72866 MITF Microphthalmia-associated transcription factor 552 A5740 AI304392 PTGFRN Prostaglandin F2 receptor negative regulator 553 A1150 NM_000560 CD53 CD53 antigen 554 A6221 N67054 RANBP5 RAN binding protein 5 555 A3680 U79751 BLZF1 Basic leucine zipper nuclear factor 1 (JEM-1) 556 A3778 BC050277 PELO Integrin, alpha 1 557 A4024 AK091336 STMN2 Stathmin-like 2 558 A4394 AF039701 MBD2 Methyl-CpG binding domain protein 2 559 A4823 D50370 NAP1L3 Nucleosome assembly protein 1-like 3 560 A6003 BC042605 FKBP5 FK506 binding protein 5 561 A0152 M19154 TGFB2 Transforming growth factor, beta 2 562 A6102 R71596 Transcribed locus 563 A0735 NM_001847 COL4A6 Collagen, type IV, alpha 6 564 A1010 D28475 CLCN6 Chloride channel 6 565 A1387 BC038588 AEBP1 AE binding protein 1 566 A1516 U24488 TNXB Tenascin XB 567 A1414 NM_001855 COL15A1 Collagen, type XV, alpha 1 568 A1815 NM_002664 PLEK Pleckstrin 569 A1951 AL833268 MEF2C MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C) 570 A2158 NM_005410 SEPP1 Selenoprotein P, plasma, 1 571 A2189 NM_000112 SLC26A2 Solute carrier family 26 (sulfate transporter), member 2 572 A2536 U48707 PPP1R1A Protein phosphatase 1, regulatory (inhibitor) subunit 1A 573 A2518 BM557396 IGFBP6 Insulin-like growth factor binding protein 6 574 A2543 NM_213674 TPM2 Tropomyosin 2 (beta) 575 A2625 L26081 SEMA3A Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A 576 A3360 NM_031850 AGTR1 Angiotensin II receptor, type 1 577 A3631 NM_005908 MANBA Mannosidase, beta A, lysosomal 578 A4641 J02854 MYL9 Myosin, light polypeptide 9, regulatory 579 A5991 BX537522 FLJ34077 Weakly similar to zinc finger protein 195 580 A1032 M87790 IGLC2 Immunoglobulin lambda constant 2 (Kern-Oz-marker) 581 A2074 CR594071 SERPINA1 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 582 A2291 AF003341 ALDH1A1 Aldehyde dehydrogenase 1 family, member A1 583 A2202 AJ001016 RAMP3 Receptor (calcitonin) activity modifying protein 3 584 A2319 AK126978 VCL Vinculin 585 A2182 CR749540 FAHD1 Hydroxyacylglutathione hydrolase 586 A2530 CA310505 APOD Apolipoprotein D 587 A2444 AY366508 LOH11CR2A Loss of heterozygosity, 11, chromosomal region 2, gene A 588 A2644 BC062476 ADH1C Alcohol dehydrogenase 1C (class I), gamma polypeptide 589 A3044 BC075840 IGHG1 Immunoglobulin heavy constant gamma 1 (G1m marker) 590 A2693 NM_002742 PRKCM Protein kinase C, mu 591 A3382 AF004021 PTGFR Prostaglandin F receptor (FP) 592 A3296 M24122 MYL3 Myosin, light polypeptide 3, alkali; ventricular, skeletal, slow 593 A3412 NM_000552 VWF Von Willebrand factor 594 A4267 BU689993 NDUFA6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14 kDa 595 A0593 NM_002290 LAMA4 Laminin, alpha 4 596 A1959 U10550 GEM GTP binding protein overexpressed in skeletal muscle 597 A2159 L10340 EEF1A2 Eukaryotic translation elongation factor 1 alpha 2 598 A2626 NM_004137 KCNMB1 Potassium large conductance calcium-activated channel, subfamily M, beta member 1 599 A3499 NM_005406 ROCK1 Rho-associated, coiled-coil containing protein kinase 1 600 A3390 BC001093 PDLIM7 PDZ and LIM domain 7 (enigma) 601 A6234 NM_000667 ADH1A Alcohol dehydrogenase 1A (class I), alpha polypeptide 602 A4545 BC056898 PLS3 Plastin 3 (T isoform) 603 A4680 NM_004517 ILK Integrin-linked kinase 604 A5306 AB046764 NBEA Neurobeachin 605 A5720 BQ787632 SPON1 Spondin 1, extracellular matrix protein 606 A0460 X55656 HBG2 Hemoglobin, gamma G 607 A0994 BC016928 OAT Ornithine aminotransferase (gyrate atrophy) 608 A1154 NM_000784 CYP27A1 Cytochrome P450, family 27, subfamily A, polypeptide 1 609 A1423 L38486 MFAP4 Microfibrillar-associated protein 4 610 A1301 AF039018 PDLIM3 PDZ and LIM domain 3 611 A1431 L43821 NEDD9 Neural precursor cell expressed, developmentally down-regulated 9 612 A1966 X81438 AMPH Amphiphysin (Stiff-Man syndrome with breast cancer 128 kDa autoantigen) 613 A2075 L02321 GSTM5 Glutathione S-transferase M5 614 A2175 J03075 PRKCSH Protein kinase C substrate 80K-H 615 A6159 T17385 Hypothetical LOC399951 616 A2557 NM_001928 DF D component of complement (adipsin) 617 A3019 J00068 ACTA1 Actin, alpha 1, skeletal muscle 618 A3511 NM_006200 PCSK5 Proprotein convertase subtilisin/kexin type 5 619 A3291 BM805032 PRSS2 Protease, serine, 2 (trypsin 2) 620 A3903 AF026692 SFRP4 Secreted frizzled-related protein 4 621 A4297 NM_012205 HAAO 3-hydroxyanthranilate 3,4- dioxygenase 622 A4403 NM_001856 COL16A1 Collagen, type XVI, alpha 1 623 A4695 NM_001003395 TPD52L1 Tumor protein D52-like 1 624 A5457 AF038193 ARL3 ADP-ribosylation factor-like 3 625 A5849 NM_024095 ASB8 Ankyrin repeat and SOCS box- containing 8 626 A0971 AY034086 DSCR1L1 Down syndrome critical region gene 1-like 1 627 A0707 NM_000677 ADORA3 Adenosine A3 receptor 628 A0745 NM_004024 ATF3 Activating transcription factor 3 629 A1510 NM_004385 CSPG2 Chondroitin sulfate proteoglycan 2 (versican) 630 A1693 X94991 ZYX Zyxin 631 A3032 NM_000055 BCHE Butyrylcholinesterase 632 A2904 BM727781 PCP4 Purkinje cell protein 4 633 A4043 NM_000304 PMP22 Peripheral myelin protein 22 634 A4136 BC035128 MXI1 MAX interactor 1 635 A4263 BX647780 ITGA5 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) 636 A4390 AB007836 TGFB1I1 Transforming growth factor beta 1 induced transcript 1 637 A5442 AF105036 KLF4 Kruppel-like factor 4 (gut) 638 A5422 W91908 GALNAC4S-6ST B cell RAG associated protein 639 A5834 AK025773 LMAN1 Lectin, mannose-binding, 1 640 A1432 L47738 CYFIP2 Cytoplasmic FMR1 interacting protein 2 641 A6243 BM564532 OPN1SW Opsin 1 (cone pigments), short- wave-sensitive (color blindness, tritan) 642 A4702 NM_014890 DOC1 Downregulated in ovarian cancer 1 643 A5428 H05313 Transcribed locus 644 A5556 BC071586 TIMP2 Tissue inhibitor of metalloproteinase 2 645 A6008 NM_005504 BCAT1 Branched chain aminotransferase 1, cytosolic 646 A1553 BC023505 ECM1 Extracellular matrix protein 1 647 A2063 U47025 PYGB Phosphorylase, glycogen; brain 648 A3015 NM_201442 C1S Complement component 1, s subcomponent 649 A3119 J04621 SDC2 Syndecan 2 (heparan sulfate proteoglycan 1, cell surface- associated, fibroglycan) 650 A3501 BC051748 TOP3A Topoisomerase (DNA) III alpha 651 A3151 M83712 CHRNA5 Cholinergic receptor, nicotinic, alpha polypeptide 5 652 A3373 L13858 SOS2 Son of sevenless homolog 2 (Drosophila) 653 A4547 NM_004586 RPS6KA3 Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 654 A5868 BC037733 SLC40A1 Solute carrier family 40 (iron- regulated transporter), member 1 655 A0578 NM_004417 DUSP1 Dual specificity phosphatase 1 656 A6152 XM_376018 KIAA1644 KIAA1644 protein 657 A3054 U01839 FY Duffy blood group 658 A3550 NM_000702 ATP1A2 ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide 659 A3299 BM696587 CRYAB Crystallin, alpha B 660 A3783 X70991 NAB2 NGFI-A binding protein 2 (EGR1 binding protein 2) 661 A4819 D17408 CNN1 Calponin 1, basic, smooth muscle 662 A4917 X83688 P2RX1 Purinergic receptor P2X, ligand- gated ion channel, 1 663 A5597 BC012347 FGF13 Fibroblast growth factor 13 664 A6010 U79271 AKT3 V-akt murine thymoma viral oncogene homolog 3 (protein kinase B, gamma) 665 A6089 CR749654 PHLDB2 Pleckstrin homology-like domain, family B, member 2 666 A0975 NM_002037 FYN FYN oncogene related to SRC, FGR, YES 667 A0875 L13740 NR4A1 Nuclear receptor subfamily 4, group A, member 1 668 A0597 X72760 LAMB2 Laminin, beta 2 (laminin S) 669 A1023 X05610 COL4A2 Collagen, type IV, alpha 2 670 A1147 NM_000129 F13A1 Coagulation factor XIII, A1 polypeptide 671 A1956 NM_004010 DMD Dystrophin (muscular dystrophy, Duchenne and Becker types) 672 A2404 M15395 ITGB2 Integrin, beta 2 (antigen CD18 (p95), lymphocyte function-associated antigen 1; macrophage antigen 1 (mac-1) beta subunit) 673 A2675 NM_005907 MAN1A1 Mannosidase, alpha, class 1A, member 1 674 A2641 X69090 MYOM1 Myomesin 1 (skelemin) 185 kDa 675 A4175 CR594469 RHOQ Ras homolog gene family, member Q 676 A4807 AJ001515 RYR3 Ryanodine receptor 3 677 A5152 AK129891 CASQ2 Calsequestrin 2 (cardiac muscle) 678 A0184 NM_000426 LAMA2 Laminin, alpha 2 (merosin, congenital muscular dystrophy) 679 A6115 NM_175709 CBX7 Chromobox homolog 7 680 A2415 M15856 LPL Lipoprotein lipase 681 A2442 AK074668 ISLR Immunoglobulin superfamily containing leucine-rich repeat 682 A2450 NM_001740 CALB2 Calbindin 2, 29 kDa (calretinin) 683 A3055 AK095384 PDE4C Phosphodiesterase 4C, cAMP- specific (phosphodiesterase E1 dunce homolog, Drosophila) 684 A3380 L20977 ATP2B2 ATPase, Ca++ transporting, plasma membrane 2 685 A3181 NM_002193 INHBB Inhibin, beta B (activin AB beta polypeptide) 686 A4053 AB005293 PLIN Perilipin 687 A4794 AF064493 LDB2 LIM domain binding 2 688 A4830 NM_004557 NOTCH4 Notch homolog 4 (Drosophila) 689 A5436 BQ009281 ELL2 Elongation factor, RNA polymerase II, 2 690 A5690 AB028952 SYNPO Synaptopodin 691 A6510 AI215810 CAPN7 Calpain 7 692 B1689 AL359062 COL8A1 Collagen, type VIII, alpha 1 693 B2439 U04735 STCH Stress 70 protein chaperone, microsome-associated, 60 kDa 694 B6764 M14338 PROS1 Protein S (alpha) 695 B8155 NM_006873 SBLF TFIIA-alpha/beta-like factor 696 A6427 BC004995 MARVELD1 MARVEL domain containing 1 697 A6436 AB014609 MRC2 Mannose receptor, C type 2 698 A7576 AI640497 C9orf103 Chromosome 9 open reading frame 103 699 A8863 BM678420 Transcribed locus 700 B1851 AA032154 FLJ22655 Hypothetical protein FLJ22655 701 B3746 AF311912 SFRP2 Secreted frizzled-related protein 2 702 B3759 AF067420 MGC27165 Hypothetical protein MGC27165 703 B4090 M34175 AP2B1 Adaptor-related protein complex 2, beta 1 subunit 704 A6545 NM_004613 TGM2 Transglutaminase 2 (C polypeptide, protein-glutamine-gamma- glutamyltransferase) 705 A6949 AB014733 SMAP-5 Golgi membrane protein SB140 706 A6923 AA677283 KIRREL Kin of IRRE like (Drosophila) 707 A9130 AF001436 CDC42EP2 CDC42 effector protein (Rho GTPase binding) 2 708 A9161 BC051700 PHF10 PHD finger protein 10 709 B0149 AF052090 NNT Nicotinamide nucleotide transhydrogenase 710 B1400 BX538213 CPEB4 Cytoplasmic polyadenylation element binding protein 4 711 B2148 M61900 712 B2723 AA018259 Full-length cDNA clone CS0DF027YN23 of Fetal brain of Homo sapiens (human) 713 A6778 M36172 MYL4 Myosin, light polypeptide 4, alkali; atrial, embryonic 714 A7349 BX647178 FILIP1 Filamin A interacting protein 1 715 B2829 AA121865 FLJ10081 Hypothetical protein FLJ10081 716 B2547 BM725055 Transcribed locus 717 A9341 BG576897 MSRB Methionine sulfoxide reductase B 718 B0275 AK092204 DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 719 B2490 BX112650 RYR2 Ryanodine receptor 2 (cardiac) 720 B2456 AL550901 CCNI Cyclin I 721 B3649 AI199480 WASF2 WAS protein family, member 2 722 B4064 NM_000047 ARSE Arylsulfatase E (chondrodysplasia punctata 1) 723 A6385 AA663484 PPP2R2B Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform 724 A8114 AL832154 CAP2 CAP, adenylate cyclase-associated protein, 2 (yeast) 725 B0739 AK074209 PLEKHA3 Pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 3 726 B0268 AK123393 CCDC3 Coiled-coil domain containing 3 727 B1032 NM_172127 CAMK2D Calcium/calmodulin-dependent protein kinase (CaM kinase) II delta 728 B4077 NM_004099 STOM Stomatin 729 B0283 AL832993 NDFIP1 Nedd4 family interacting protein 1 730 B2130 NM_000448 RAG1 Recombination activating gene 1 731 B2457 NM_007353 GNA12 Guanine nucleotide binding protein (G protein) alpha 12 732 B4085 NM_198098 AQP1 Aquaporin 1 (channel-forming integral protein, 28 kDa) 733 B4092 AB011126 FNBP1 Formin binding protein 1 734 B6287 U66680 735 A7286 NM_021201 MS4A7 Membrane-spanning 4-domains, subfamily A, member 7 736 A8203 AK026966 AK3 Adenylate kinase 3 737 A7795 BC044582 UBL3 Ubiquitin-like 3 738 A8531 BX537531 FBLN5 Fibulin 5 739 B1081 AK096303 FLJ38984 Hypothetical protein FLJ38984 740 B1985 AI052390 FLJ20071 Dymeclin 741 B4230 AK054596 IGBP1 Immunoglobulin (CD79A) binding protein 1 742 B4078 AK093049 SERPINA3 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 743 C0670 AK093067 CHPT1 Choline phosphotransferase 1 744 A6409 AK091288 C9orf19 Chromosome 9 open reading frame 19 745 A6664 H19830 DKFZP434G156 Hypothetical protein DKFZp434G156 746 A7411 BC035028 SERPIND1 Serine (or cysteine) proteinase inhibitor, clade D (heparin cofactor), member 1 747 A7145 X52005 748 A7963 AK024964 NFIA KIAA0485 protein 749 A7972 NM_170677 MEIS2 Meis1, myeloid ecotropic viral integration site 1 homolog 2 (mouse) 750 A8433 NM_005843 STAM2 Signal transducing adaptor molecule (SH3 domain and ITAM motif) 2 751 B2609 AL833069 KIAA1434 Hypothetical protein KIAA1434 752 A9305 BC035417 Transcribed locus, weakly similar to NP_650255.1 Drosophila melanogaster CG11670 gene 753 A9187 BC034222 HRLP5 H-rev107-like protein 5 754 A9801 AI350750 PDGFD DNA-damage inducible protein 1 755 B1288 H73979 CACNB2 Calcium channel, voltage-dependent, beta 2 subunit 756 B2506 AL834231 MTPN Myotrophin 757 B4086 NM_006206 PDGFRA Platelet-derived growth factor receptor, alpha polypeptide 758 A6672 H27000 WBSCR17 Williams-Beuren syndrome chromosome region 17 759 A6683 AB088477 PER1 Period homolog 1 (Drosophila) 760 A8568 BQ071428 CUEDC2 CUE domain containing 2 761 A9468 BX110596 Homo sapiens, clone IMAGE: 4799216, mRNA 762 B8113 BC020848 RNASE6 Ribonuclease, RNase A family, k6 763 B6773 BC077077 DPYSL3 Dihydropyrimidinase-like 3 764 A6781 CB529051 G0S2 Putative lymphocyte G0/G1 switch gene 765 A6410 XM_496907 PEG10 Paternally expressed 10 766 A6665 AW450890 LMO3 LIM domain only 3 (rhombotin-like 2) 767 A6936 AI766077 FLJ13456 Hypothetical protein FLJ13456 768 A9993 AB007903 GPRASP1 G protein-coupled receptor associated sorting protein 1 769 B0327 NM_144658 DOCK11 Dedicator of cytokinesis 11 770 B4288 AK092766 OLFML3 Olfactomedin-like 3 771 A6776 BM726594 COX7A1 Cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) 772 A7599 AK095147 CDNA FLJ37828 fis, clone BRSSN2006575 773 A7330 NM_018434 RNF130 Ring finger protein 130 774 A9462 BM474898 SLIT2 Slit homolog 2 (Drosophila) 775 A9042 NM_022349 MS4A6A Membrane-spanning 4-domains, subfamily A, member 6A 776 A6522 BC045177 FLJ30046 Hypothetical protein FLJ30046 777 A6530 NM_006988 ADAMTS1 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 778 A6567 AK096428 PDK4 Pyruvate dehydrogenase kinase, isoenzyme 4 779 A7663 BX647421 FSTL1 Follistatin-like 1 780 B2584 CR620669 PBX3 Pre-B-cell leukemia transcription factor 3 781 A8129 AB067468 KIAA1881 KIAA1881 782 A8552 AK056721 LOC56181 Hypothetical protein RP1-317E23 783 A8591 BC078139 EIF2C2 Eukaryotic translation initiation factor 2C, 2 784 A8744 NM_001233 CAV2 Caveolin 2 785 B0328 AK094236 DDIT4L DNA-damage-inducible transcript 4- like 786 B4847 AA490011 LTBP1 Latent transforming growth factor beta binding protein 1 787 A6611 N58556 DKFZp547K1113 Hypothetical protein DKFZp547K1113 788 A6317 AI205684 HSPA2 Heat shock 70 kDa protein 2 789 A7191 BC007655 PPP1R2 Protein phosphatase 1, regulatory (inhibitor) subunit 2 790 A8600 CR749355 hIAN2 Human immune associated nucleotide 2 791 B1649 AA244092 Chromosome 9 pericentromeric mRNA sequence 792 B4691 NM_024605 ARHGAP10 Rho GTPase activating protein 10 793 B6518 CA419435 GNPDA2 Glucosamine-6-phosphate deaminase 2 794 B9201 BX647427 WIF1 WNT inhibitory factor 1 795 A6458 AK127289 SLCO2B1 Solute carrier organic anion transporter family, member 2B1 796 A6602 W87690 ITGA9 Integrin, alpha 9 797 A6863 AK027199 MGC48972 Hypothetical protein MGC48972 798 A6906 BC050423 TMEM22 Transmembrane protein 22 799 A8147 AY422170 TP53INP2 Tumor protein p53 inducible nuclear protein 2 800 B4154 D87074 RIMS3 Regulating synaptic membrane exocytosis 3 801 B6579 AK126500 APEG1 Aortic preferentially expressed protein 1 802 A6712 NM_182643 DLC1 Deleted in liver cancer 1 803 A6993 H10356 CDNA FLJ36544 fis, clone TRACH2006378 804 A7225 NM_002729 HHEX Hematopoietically expressed homeobox 805 A6837 BX412783 LOC283140 Hypothetical protein LOC283140 806 A7425 NM_003250 THRA Thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb- a) oncogene homolog, avian) 807 A8162 AL832955 TNFAIP9 Tumor necrosis factor, alpha-induced protein 9 808 A7679 M97675 ROR1 Receptor tyrosine kinase-like orphan receptor 1 809 B2084 S45018 CHAT Choline acetyltransferase 810 A6593 AF007150 ANGPTL2 Angiopoietin-like 2 811 A7075 NM_004982 KCNJ8 Potassium inwardly-rectifying channel, subfamily J, member 8 812 A7710 AK125609 CKIP-1 CK2 interacting protein 1; HQ0024c protein 813 A7772 BC028314 SURF1 Surfeit 1 814 A9120 AF332010 CDV-1 Carnitine deficiency-associated gene expressed in ventricle 1 815 B0202 NM_021914 CFL2 Cofilin 2 (muscle) 816 B0240 AA081184 TCF4 Transcription factor 4 817 B5402 XM_375377 KIAA0513 KIAA0513 818 B8811 D86962 GRB10 Growth factor receptor-bound protein 10 819 A6613 AB018278 SV2B Synaptic vesicle glycoprotein 2B 820 A6583 XM_371114 FHOD3 Formin homology 2 domain containing 3 821 A6876 AA705804 DPT Dermatopontin 822 A7426 BG617617 SAA2 Serum amyloid A2 823 A7689 X00457 HLA-DPA1 Major histocompatibility complex, class II, DP alpha 1 824 A8605 AK025205 DKFZP564O0823 DKFZP564O0823 protein 825 A8639 AI368204 ENPP3 Ectonucleotide pyrophosphatase/phosphodiesterase 3 826 A8647 XM_290734 Similar to ataxin 2-binding protein 1 isoform 4; hexaribonucleotide binding protein 1 827 A9250 BC062575 RHOJ Ras homolog gene family, member J 828 B3940 K02765 C3 Complement component 3 829 B3977 AI056268 PARVA Parvin, alpha 830 B4699 NM_003450 ZNF174 Zinc finger protein 174 831 B8782 AK022926 CTNNAL1 Catenin (cadherin-associated protein), alpha-like 1 832 A6719 AI302184 SQRDL Sulfide quinone reductase-like (yeast) 833 A7464 AF081287 CTDP1 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) phosphatase, subunit 1 834 A7246 N75862 EYA4 Eyes absent homolog 4 (Drosophila) 835 A7773 NM_002504 NFX1 Nuclear transcription factor, X-box binding 1 836 A8155 CD242398 LOC51255 Hypothetical protein LOC51255 837 B2641 BX094063 PIN4 Protein (peptidyl-prolyl cis/trans isomerase) NIMA-interacting, 4 (parvulin) 838 A9121 AB002388 ZNF536 Zinc finger protein 536 839 A9280 AW136599 HUNK Hormonally upregulated Neu- associated kinase 840 B0241 BC056414 PLVAP Plasmalemma vesicle associated protein 841 B1531 BC063304 NPR1 Natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptorA) 842 B2663 BC009978 ACTC Actin, alpha, cardiac muscle 843 B4213 NM_001001937 ATP5A1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle 844 A6348 AK026653 C14orf168 Chromosome 14 open reading frame 168 845 A6842 AB043585 RPRM Reprimo, TP53 dependant G2 arrest mediator candidate 846 A8761 BM984852 C6orf166 Hypothetical protein PRO2266 847 B0337 R28608 MAPRE2 Microtubule-associated protein, RP/EB family, member 2 848 B5155 W84893 AGTRL1 Angiotensin II receptor-like 1 849 B7659 AF541281 LPPR4 Plasticity related gene 1 850 B6542 NM_014819 PJA2 Praja 2, RING-H2 motif containing 851 A7775 BC033820 FGL2 Fibrinogen-like 2 852 A8156 BQ010373 HEG HEG homolog 853 B2659 AI025259 Transcribed locus 854 A9282 AF086912 OGN Osteoglycin (osteoinductive factor, mimecan) 855 B0845 H68305 PRKAB2 Protein kinase, AMP-activated, beta 2 non-catalytic subunit 856 B3924 AK075151 HSPB7 Heat shock 27 kDa protein family, member 7 (cardiovascular) 857 B4008 XM_167709 C10orf38 Chromosome 10 open reading frame 38 858 C4095 NM_002122 HLA-DQA1 Major histocompatibility complex, class II, DQ alpha 1 859 B9009 XM_039796 TNIK TRAF2 and NCK interacting kinase 860 A6475 BC032508 FLJ10781 Hypothetical protein FLJ10781 861 A6358 AK056079 JAM2 Junctional adhesion molecule 2 862 A7230 NM_001845 COL4A1 Collagen, type IV, alpha 1 863 A9373 AK128695 COL6A2 Collagen, type VI, alpha 2 864 A9103 AK091635 FLJ11200 Hypothetical protein FLJ11200 865 A9081 BC000693 ACTR1A ARP1 actin-related protein 1 homolog A, centractin alpha (yeast) 866 A9381 AL117605 CDNA: FLJ21418 fis, clone COL04072 867 A9719 XM_294521 FLJ43950 FLJ43950 protein 868 B1004 NM_004530 MMP2 Matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) 869 B4674 AA149429 ATP10D ATPase, Class V, type 10D 870 A6856 XM_051081 TBC1D12 TBC1 domain family, member 12 871 A7467 BC034989 P2RY14 Purinergic receptor P2Y, G-protein coupled, 14 872 A7088 AB031046 TCF7L1 Transcription factor 7-like 1 (T-cell specific, HMG-box) 873 A7222 NM_001911 CTSG Cathepsin G 874 A7893 AA417560 Transcribed locus 875 A8030 AL137734 DKFZp586C0721 Hypothetical protein DKFZp586C0721 876 A8514 AL110212 H2AFV H2A histone family, member V 877 A9870 AB040938 KIAA1505 KIAA1505 protein 878 B1676 BC025985 IGHG4 Immunoglobulin heavy constant gamma 4 (G4m marker) 879 A6447 AK127088 EPB41L2 Erythrocyte membrane protein band 4.1-like 2 880 A6457 AJ318805 CDNA FLJ44429 fis, clone UTERU2015653 881 A6360 AL390127 KLF13 Kruppel-like factor 13 882 A7000 XM_496727 DKFZP564J102 DKFZP564J102 protein 883 A6844 AL831898 LOC285812 Hypothetical protein LOC285812 884 A7239 AA523541 DSIPI Delta sleep inducing peptide, immunoreactor 885 A8482 BC047492 ADHFE1 Alcohol dehydrogenase, iron containing, 1 886 A8493 AA780301 CTSF Cathepsin F 887 A9564 NM_000076 CDKN1C Cyclin-dependent kinase inhibitor 1C (p57, Kip2) 888 B2874 AA883488 KIAA0408 KIAA0408 889 B6561 AB014544 KIAA0644 KIAA0644 gene product 890 B7552 NM_016143 NSFL1C NSFL1 (p97) cofactor (p47) 891 A6751 NM_002258 KLRB1 Killer cell lectin-like receptor subfamily B, member 1 892 A8186 BM551020 SCAMP2 Secretory carrier membrane protein 2 893 A8159 BX537904 APG5L APG5 autophagy 5-like (S. cerevisiae) 894 A8823 N26005 PPP1R3C Protein phosphatase 1, regulatory (inhibitor) subunit 3C 895 B4810 BM701072 KIAA0103 KIAA0103 896 B9056 AF433662 ARHGEF3 Rho guanine nucleotide exchange factor (GEF) 3 897 B2978 AA442090 FLJ10292 Hypothetical protein FLJ10292 898 B3794N BC033829 AKAP12 A kinase (PRKA) anchor protein (gravin) 12 899 B3766 NM_000933 PLCB4 Phospholipase C, beta 4 900 B4194N BG292094 FLJ11000 Hypothetical protein FLJ11000 901 B6051 R32860 MOBKL2B MOB1, Mps One Binder kinase activator-like 2B (yeast) 902 B6511 AK124739 CDNA FLJ36725 fis, clone UTERU2012230 903 B6358 AL161983 MGC39820 Hypothetical protein MGC39820 904 B7880N AK125119 C6orf68 Chromosome 6 open reading frame 68 905 B9198 AK123132 MSRA Methionine sulfoxide reductase A 906 B8754 AL833264 FEM1B Fem-1 homolog b (C. elegans) 907 B9564 CR609948 KPNB1 Karyopherin (importin) beta 1 908 B9394 AL117521 C20orf77 Chromosome 20 open reading frame 77 909 A1871N NM_198235 RNASE1 Ribonuclease, RNase A family, 1 (pancreatic) 910 A3439N BM994174 HBB Hemoglobin, beta 911 A7605 R15801 NRN1 Neuritin 1 912 B4394 N46424 RAI14 Retinoic acid induced 14 913 B3827 N20989 ANTXR1 Anthrax toxin receptor 1 914 B4574 AK160376 FLJ12895 Hypothetical protein FLJ12895 915 B6373 BX423161 LHPP Phospholysine phosphohistidine inorganic pyrophosphate phosphatase 916 B7122 AA480009 CDNA FLJ13569 fis, clone PLACE1008369 917 B7310 R72837 DKFZP434F2021 DKFZP434F2021 protein 918 B8308 NM_001001936 KIAA1914 KIAA1914 919 B7903 N49237 Homo sapiens, clone IMAGE: 5312516, mRNA 920 B8141 BC042478 DKFZP434F0318 Hypothetical protein DKFZp434F0318 921 B9647 AK125651 FLJ43663 Hypothetical protein FLJ43663 922 A0925N L42374 PPP2R5B Protein phosphatase 2, regulatory subunit B (B56), beta isoform 923 A6533N AL833076 FLJ14281 Hypothetical protein FLJ14281 924 A6272 NM_139346 BIN1 Bridging integrator 1 925 B4133 BC039740 LOC84549 RNA binding protein 926 B4694 AK074046 ZNF521 Zinc finger protein 521 927 B5489 NM_003916 AP1S2 Adaptor-related protein complex 1, sigma 2 subunit 928 B6306 AF107454 C7orf2 Chromosome 7 open reading frame 2 929 B7500 H14059 LOC197336 Similar to RIKEN cDNA 3230401M21 [Mus musculus] 930 B8069 NM_013366 ANAPC2 Anaphase promoting complex subunit 2 931 B8422 CA310622 ACTR3 ARP3 actin-related protein 3 homolog (yeast) 932 B8679 NM_030569 ITIH5 Inter-alpha (globulin) inhibitor H5 933 B9427 BC047114 CDNA clone IMAGE: 5313062, partial cds 934 B9435 Z39318 SP2 Sp2 transcription factor 935 A4798N NM_174953 ATP2A3 ATPase, Ca++ transporting, ubiquitous 936 A0774N BC012613 CPA3 Carboxypeptidase A3 (mast cell) 937 A6640N BC001816 RAP1GDS1 RAP1, GTP-GDP dissociation stimulator 1 938 A8783N AA621565 ENPP1 Ectonucleotide pyrophosphatase/phosphodiesterase 1 939 B0870N CR610395 ASF1A ASF1 anti-silencing function 1 homolog A (S. cerevisiae) 940 B5768 BX647147 Transcribed locus, weakly similar to XP_375099.1 hypothetical protein LOC283585 [Homo sapiens] 941 B6365 AK091292 FATJ Fat-like cadherin FATJ 942 B6571 BC039332 LOC285086 Hypothetical protein LOC285086 943 B7170N NM_019035 PCDH18 Protocadherin 18 944 B9172 AK092542 C2orf32 Chromosome 2 open reading frame 32 945 B9634 BX110180 Transcribed locus 946 B9317 N24737 Transcribed locus 947 B9419 BM996307 LNX Ligand of numb-protein X 948 A2660N NM_021023 CFHL3 Complement factor H-related 3 949 A0881N Z21707 ZNF197 Zinc finger protein 197 950 A1816N BC075800 PRKAR2B Protein kinase, cAMP-dependent, regulatory, type II, beta 951 A0702N BQ189297 FLT1 Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) 952 A0936N M86852 PXMP3 Peroxisomal membrane protein 3, 35 kDa (Zellweger syndrome) 953 B4137 NM_053025 MYLK Myosin, light polypeptide kinase 954 B4949 R26358 SLMAP Sarcolemma associated protein 955 B5172N NM_001289 CLIC2 Chloride intracellular channel 2 956 B6700 AL133579 STARD9 START domain containing 9 957 B6104N BM987057 KIAA0563 KIAA0563 gene product 958 B7105 AK055782 PDLIM2 PDZ and LIM domain 2 (mystique) 959 B7930 N21096 960 B7887 BU580616 FLJ10159 Hypothetical protein FLJ10159 961 B8081 BM981462 FLJ13710 Hypothetical protein FLJ13710 962 B8438 AA403307 UBE4B Ubiquitination factor E4B (UFD2 homolog, yeast) 963 B9300 CA446432 C6orf66 Chromosome 6 open reading frame 66 964 A6448N AK127801 FLJ46603 FLJ46603 protein 965 A9346N AY358379 PP2135 PP2135 protein 966 B3779 BF966783 967 B4396 W58589 DDR2 Discoidin domain receptor family, member 2 968 B4568 AK021950 PRTFDC1 Phosphoribosyl transferase domain containing 1 969 B4956 NM_005737 ARL7 ADP-ribosylation factor-like 7 970 B4577 AY081219 ABCC4 ATP-binding cassette, sub-family C (CFTR/MRP), member 4 971 B5199 AK098381 ADCY5 Adenylate cyclase 5 972 B5399 XM_056455 D2S448 Melanoma associated gene 973 B6366 AK130263 KIAA1430 KIAA1430 974 B7304N AA777308 C6orf60 Chromosome 6 open reading frame 60 975 B7312 BU738244 Hypothetical gene supported by AK094796 976 B7171 H75419 CYBRD1 Cytochrome b reductase 1 977 B7708 AA938297 FLJ20716 Hypothetical protein FLJ20716 978 B8098 R42864 PAPOLA Poly(A) polymerase alpha 979 B7768 BC017032 GCNT3 Glucosaminyl (N-acetyl) transferase 3, mucin type 980 B8770 J04605 PEPD Peptidase D 981 B9603 BM679454 ASAM Adipocyte-specific adhesion molecule 982 B9803 AF414088 COL21A1 Collagen, type XXI, alpha 1 983 A4655N NM_001164 APBB1 Amyloid beta (A4) precursor protein-binding, family B, member 1 (Fe65) 984 A1818N NM_033138 CALD1 Caldesmon 1 985 A3200N AK122763 COL5A1 Collagen, type V, alpha 1 986 B2864 AI088622 PRKCDBP Protein kinase C, delta binding protein 987 B4348 AK055071 PIGK Phosphatidylinositol glycan, class K 988 B8670 NM_021038 MBNL1 Muscleblind-like (Drosophila) 989 B9836 R79561 ARRDC3 Arrestin domain containing 3 990 A1779N AF025534 LILRB5 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 5 991 A3034N BC027913 PPP3R1 Protein phosphatase 3 (formerly 2B), regulatory subunit B, 19 kDa, alpha isoform (calcineurin B, type I) 992 A3147N U20938 DPYD Dihydropyrimidine dehydrogenase 993 A1963N BC047756 QPCT Glutaminyl-peptide cyclotransferase (glutaminyl cyclase) 994 B3829 AF091434 PDGFC Platelet derived growth factor C 995 B4578 AI290343 STC2 Stanniocalcin 2 996 B5151 BU627644 7h3 Hypothetical protein FLJ13511 997 B4614 AL833852 TAZ Transcriptional co-activator with PDZ-binding motif (TAZ) 998 B4971 NM_020443 NAV1 Neuron navigator 1 999 B6319 BX414085 ICSBP1 Interferon consensus sequence binding protein 1 1000 B7526 R40594 CYP2U1 Cytochrome P450, family 2, subfamily U, polypeptide 1 1001 B9790 BC067746 CLEC1 C-type lectin-like receptor-1 1002 B9341 BC012984 ALS2CR19 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 19 1003 A0907N NM_016083 CNR1 Cannabinoid receptor 1 (brain) 1004 A5065 BC036661 CMKOR1 Chemokine orphan receptor 1 1005 A3369N L13283 MUC7 Mucin 7, salivary 1006 A8391N AA482082 FOXK1 Forkhead box K1 1007 B4364 CD365397 TRPV2 Transient receptor potential cation channel, subfamily V, member 2 1008 B3958 AF145713 SCHIP1 Schwannomin interacting protein 1 1009 B5164 R37342 PARVG Parvin, gamma 1010 B6108 AW409897 LOC91461 Hypothetical protein BC007901 1011 B6565N BC007997 RERG RAS-like, estrogen-regulated, growth inhibitor 1012 B7875 H17818 Transcribed locus 1013 B9007 BQ028161 hSyn Brain synembryn 1014 A1780N CR606785 ENPP2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin) 1015 A1151N M55618 TNC Tenascin C (hexabrachion) 1016 A6369N NM_013374 PDCD6IP Programmed cell death 6 interacting protein 1017 B3930 XM_290629 C14orf78 Chromosome 14 open reading frame 78 1018 B4217 BU608626 WFDC2 WAP four-disulfide core domain 2 1019 B4447 NM_032287 LDOC1L Leucine zipper, down-regulated in cancer 1-like 1020 B5721N AK024116 FLJ14054 Hypothetical protein FLJ14054 1021 B5949 NM_016293 BIN2 Bridging integrator 2 1022 B6719 BX537713 Homo sapiens, clone IMAGE: 4150640, mRNA 1023 B6738 BX640757 DRCTNNB1A Down-regulated by Ctnnb1, a 1024 A1669 M95787 TAGLN Transgelin 1025 A0919N J05550 MRC1 Mannose receptor, C type 1 1026 A2753N BC009924 NPTX2 Neuronal pentraxin II 1027 A0969N NM_001873 CPE Carboxypeptidase E 1028 B3911 BC038457 DKFZP586H2123 Regeneration associated muscle protease 1029 B4953 AB007960 SH3GLB1 SH3-domain GRB2-like endophilin B1 1030 B5382N AK125194 MAP1B Microtubule-associated protein 1B 1031 B6568 AK091271 GPR161 G protein-coupled receptor 161 1032 B7877 AB029033 IQSEC3 IQ motif and Sec7 domain 3 1033 B9777 NM_030781 COLEC12 Collectin sub-family member 12 1034 A3079 J04599 BGN Biglycan 1035 A3160N NM_000125 ESR1 Estrogen receptor 1 1036 A1981 U58514 CHI3L2 Chitinase 3-like 2 1037 A6696 NM_012072 C1QR1 Complement component 1, q subcomponent, receptor 1 1038 A8809N NM_000755 CRAT Carnitine acetyltransferase 1039 B1090N AF361473 ROBO4 Roundabout homolog 4, magic roundabout (Drosophila) 1040 B3933 AY358360 ELTD1 EGF, latrophilin and seven transmembrane domain containing 1 1041 B3966 BC047724 C10orf128 Chromosome 10 open reading frame 128 1042 B3831 AK125356 KLHL13 Kelch-like 13 (Drosophila) 1043 B4450 BC048969 TSPYL1 TSPY-like 1 1044 B5396 AF208863 C6orf209 Chromosome 6 open reading frame 209 1045 B5410 BC033183 CHST3 Carbohydrate (chondroitin 6) sulfotransferase 3 1046 B6082 BX537781 FNDC5 Fibronectin type III domain containing 5 1047 B7003N AF045584 SLC43A1 Solute carrier family 43, member 1 1048 B7922 NM_181844 BCL6B B-cell CLL/lymphoma 6, member B (zinc finger protein) 1049 B8790 AK123915 ZBED3 Zinc finger, BED domain containing 3 1050 A0055N AF058925 JAK2 Janus kinase 2 (a protein tyrosine kinase) 1051 A7633 AL136578 MGC3040 Hypothetical protein MGC3040 1052 B5842N AF545852 MK2S4 Protein kinase substrate MK2S4 1053 B7363 AL832469 Hypothetical gene supported by BX647608 1054 B9053 AB023158 RAB11FIP2 RAB11 family interacting protein 2 (class I) 1055 A0560N NM_000618 IGF1 Insulin-like growth factor 1 (somatomedin C) 1056 A1624N NM_003034 SIAT8A Sialyltransferase 8A (alpha-N- acetylneuraminate: alpha-2,8- sialyltransferase, GD3 synthase) 1057 A7760N BC047390 ARID5A AT rich interactive domain 5A (MRF1-like) 1058 A8879N CR597401 HCA112 Hepatocellular carcinoma-associated antigen 112 1059 A5504N AK056479 SPRED2 Sprouty-related, EVH1 domain containing 2 1060 B2957 CR599541 TFIP11 Tuftelin interacting protein 11 1061 B3586 AA748009 PPP2R5E Protein phosphatase 2, regulatory subunit B (B56), epsilon isoform 1062 B4320 N56931 C5orf4 Chromosome 5 open reading frame 4 1063 B4277 AJ420529 STX7 Syntaxin 7 1064 B3893 AY549722 ITLN1 Intelectin 1 (galactofuranose binding) 1065 B7204N AK074765 CA14 Carbonic anhydrase XIV 1066 B7444 AW452608 C9orf87 Chromosome 9 open reading frame 87 1067 B8593 BU624282 KIAA0779 KIAA0779 protein 1068 B9132 AA455877 MRVI1 Murine retrovirus integration site 1 homolog 1069 B9504 AA521163 PTEN Phosphatase and tensin homolog (mutated in multiple advanced cancers 1) 1070 A8864N N93511 FLJ10853 Hypothetical protein FLJ10853 1071 B2559 CA426475 HBE1 Hemoglobin, epsilon 1 1072 B4235N AK095908 MFGE8 Milk fat globule-EGF factor 8 protein 1073 B4291 AK025198 XIST X (inactive)-specific transcript 1074 B4245 AF034176 NUDT3 Nudix (nucleoside diphosphate linked moiety X)-type motif 3 1075 B7559 AB073386 SGEF Src homology 3 domain-containing guanine nucleotide exchange factor 1076 B8203 NM_018325 C9orf72 Chromosome 9 open reading frame 72 1077 B8035 AL834240 KIAA1576 KIAA1576 protein 1078 B8212 AK023159 LSM11 LSM11, U7 small nuclear RNA associated 1079 A0085N D37965 PDGFRL Platelet-derived growth factor receptor-like 1080 A4391N NM_003155 STC1 Stanniocalcin 1 1081 A1437N NM_002210 ITGAV Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) 1082 A7247N AL133118 EMCN Endomucin 1083 A8289N AK127420 Transcribed locus, weakly similar to XP_375268.2 similar to FLJ43276 protein [Homo sapiens] 1084 B3883 BC027937 RAI2 Retinoic acid induced 2 1085 B4321 BX648583 EDIL3 EGF-like repeats and discoidin I-like domains 3 1086 B4684 BC036485 Homo sapiens, clone IMAGE: 5261213, mRNA 1087 B5442 AK124604 LOC283537 Hypothetical protein LOC283537 1088 B6492 AK057151 CDNA FLJ32589 fis, clone SPLEN2000443 1089 B7082 AK055323 CDNA clone IMAGE: 5263177, partial cds 1090 B9505 NM_004796 NRXN3 Neurexin 3 1091 A3538 J03464 COL1A2 Collagen, type I, alpha 2 1092 A7704N NM_003749 IRS2 Insulin receptor substrate 2 1093 B3059 NM_004755 RPS6KA5 Ribosomal protein S6 kinase, 90 kDa, polypeptide 5 1094 B3834 AB033040 RNF150 Ring finger protein 150 1095 B4237 XM_290941 PRNPIP Prion protein interacting protein 1096 B4498N BX648979 SLC41A1 Solute carrier family 41, member 1 1097 B4661 AI765053 PTPRD Protein tyrosine phosphatase, receptor type, D 1098 B4633 AL162008 CLIC4 Chloride intracellular channel 4 1099 B5019 BQ574410 Full-length cDNA clone CS0DI014YH21 of Placenta Cot 25- normalized of Homo sapiens (human) 1100 B7429 BM723215 SMARCE1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1 1101 B8028 AK057742 C10orf46 Chromosome 10 open reading frame 46 1102 B8036 R20340 ATP5S ATP synthase, H+ transporting, mitochondrial F0 complex, subunit s (factor B) 1103 B8213 AA729769 LOC112476 Similar to lymphocyte antigen 6 complex, locus G5B; G5b protein; open reading frame 31 1104 B9470 N29574 RRAGD Ras-related GTP binding D 1105 A2632N NM_003816 ADAM9 A disintegrin and metalloproteinase domain 9 (meltrin gamma) 1106 B0081N AB040962 KIAA1529 KIAA1529 1107 B3699 NM_006617 NES Nestin 1108 B8411 BX412247 EFHD1 EF hand domain containing 1 1109 A2019N AA442410 EMP1 Epithelial membrane protein 1 1110 B4240 BC018652 FXYD6 FXYD domain containing ion transport regulator 6 1111 B4249 BC070071 RBM16 RNA binding motif protein 16 1112 B3721 AB023177 KIAA0960 KIAA0960 protein 1113 B5418 BQ573990 ZNF148 Zinc finger protein 148 (pHZ-52) 1114 B5464 AK127355 SEC23A Sec23 homolog A (S. cerevisiae) 1115 B7185 W61217 RAB23 RAB23, member RAS oncogene family 1116 B7996N BQ445850 CDNA clone IMAGE: 5561426, partial cds 1117 B8341 BC043003 NEK7 NIMA (never in mitosis gene a)- related kinase 7 1118 B8547 BC033746 PNCK Pregnancy upregulated non- ubiquitously expressed CaM kinase 1119 B8351 R26919 DSCR1L2 Down syndrome critical region gene 1-like 2 1120 B8389 AK095203 PDE3A Phosphodiesterase 3A, cGMP- inhibited 1121 B9722 BQ773658 Hypothetical LOC402560 1122 A1600N NM_001844 COL2A1 Collagen, type II, alpha 1 (primary osteoarthritis, spondyloepiphyseal dysplasia, congenital) 1123 A1471N M83772 FMO3 Flavin containing monooxygenase 3 1124 A1634N BC026324 MMD Monocyte to macrophage differentiation-associated 1125 A2633N BX648814 ANGPT1 Angiopoietin 1 1126 B4030 AK055793 C20orf129 Chromosome 20 open reading frame 129 1127 B4339 W73738 TMEM25 Transmembrane protein 25 1128 B4891 W19216 PKIG Protein kinase (cAMP-dependent, catalytic) inhibitor gamma 1129 B5059N T88953 Transcribed locus 1130 B6284 AK096240 Similar to protein of fungal metazoan origin like (11.1 kD) (2C514) 1131 B7814 BC039414 Homo sapiens, clone IMAGE: 5302158, mRNA 1132 B8234 AF070632 Clone 24405 mRNA sequence 1133 B8924 AI357442 SPARC Secreted protein, acidic, cysteine- rich (osteonectin) 1134 A0038N W73825 TCF21 Transcription factor 21 1135 B3584 AA917358 Transcribed locus 1136 B6250 N30317 LOC91526 Hypothetical protein DKFZp434D2328 1137 B7018 R00826 RAB3GAP RAB3 GTPase-ACTIVATING PROTEIN 1138 B7435 AK093246 RPL13 Ribosomal protein L13 1139 B8344 AB019210 PGM2L1 Phosphoglucomutase 2-like 1 1140 B8850 Z30256 KIF1B Kinesin family member 1B 1141 A2087N BC012617 ACTG2 Actin, gamma 2, smooth muscle, enteric 1142 A4459N BC013188 TPST1 Tyrosylprotein sulfotransferase 1 1143 A7346N N70296 ANK3 Ankyrin 3, node of Ranvier (ankyrin G) 1144 B3695 BC017312 MGC3047 Hypothetical protein MGC3047 1145 B3889 BC013042 MGC7036 Hypothetical protein MGC7036 1146 B4032 AF545571 SULF1 Sulfatase 1 1147 B5618 AA502764 RKHD1 Ring finger and KH domain containing 1 1148 B6405 AA045332 ME1 Malic enzyme 1, NADP(+)- dependent, cytosolic 1149 B6035N AF205632 SH3BP3 SH3-domain binding protein 3 1150 B7441 AA994299 C16orf30 Chromosome 16 open reading frame 30 1151 B7274 BM671249 BAZ2A Bromodomain adjacent to zinc finger domain, 2A 1152 B8865 N66810 Homo sapiens, clone IMAGE: 4690669, mRNA 1153 B8404 AF173389 EEA1 Early endosome antigen 1, 162 kD 1154 A4381N U81523 EBAF Endometrial bleeding associated factor (left-right determination, factor A; transforming growth factor beta superfamily) 1155 A6909 NM_018667 SMPD3 Sphingomyelin phosphodiesterase 3, neutral membrane (neutral sphingomyelinase II) 1156 A7232N BX648421 IGJ Immunoglobulin J polypeptide, linker protein for immunoglobulin alpha and mu polypeptides 1157 B4665N AA045171 1158 B4638 BX648635 LIFR Leukemia inhibitory factor receptor 1159 B5081N AL832416 C9orf13 Chromosome 9 open reading frame 13 1160 B5460 BX537492 FLJ23091 Putative NFkB activating protein 373 1161 B5427 CR600360 DNAJA2 DnaJ (Hsp40) homolog, subfamily A, member 2 1162 B5292 BQ574739 SMAP Small acidic protein 1163 B7193N BX109986 Transcribed locus 1164 B7424 CA503060 FLJ21069 Hypothetical protein FLJ21069 1165 B8040 AJ420553 ID4 Inhibitor of DNA binding 4, dominant negative helix-loop-helix protein 1166 B8354 NM_003387 WASPIP Wiskott-Aldrich syndrome protein interacting protein 1167 B8628 AA658236 HECTD2 HECT domain containing 2 1168 B9368 AF504647 Cilia-associated protein (CYS1) 1169 B9749 BQ575959 HTRA3 Serine protease HTRA3 1170 A1397N AK091875 PPP2CB Protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform 1171 A1485N BC050283 WASF3 WAS protein family, member 3 1172 A6126N H11384 CDC42EP3 CDC42 effector protein (Rho GTPase binding) 3 1173 B4330 AB020637 KIAA0830 KIAA0830 protein 1174 B4491 BX537444 ATP2B4 ATPase, Ca++ transporting, plasma membrane 4 1175 B5815 T72611 Transcribed locus 1176 B6819N AL542335 EEF1A1 Eukaryotic translation elongation factor 1 alpha 1 1177 B7058 BX094037 Transcribed locus 1178 B8051 AK094950 CDNA FLJ37631 fis, clone BRCOC2015944 1179 B8866 CR749586 FLJ11088 GGA binding partner 1180 B8599 R37079 PDZRN4 PDZ domain containing RING finger 4 1181 B9502 AL050227 PTGER3 Prostaglandin E receptor 3 (subtype EP3) 1182 C0213 BX110085 Transcribed locus 1183 C0226 AK074924 KIAA0853 KIAA0853 1184 C3653 BC066956 VIM Vimentin 1185 C4908 BX118828 Transcribed locus 1186 C6830 R49122 FLJ14800 Hypothetical protein FLJ14800 1187 C7111 T15991 Transcribed locus 1188 C8074 X79204 ATXN1 Ataxin 1 1189 C9718 W94051 DTNA Dystrobrevin, alpha 1190 C0685 H27764 SLC18A2 Solute carrier family 18 (vesicular monoamine), member 2 1191 C3767 BC018128 FGFR1 Fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome) 1192 C4060 N35250 1193 C7466 NM_003480 MFAP5 Microfibrillar associated protein 5 1194 C8953 AL136678 DEPDC6 DEP domain containing 6 1195 C9351 AA195210 DKFZP761M1511 Hypothetical protein DKFZP761M1511 1196 C3791 BU170801 PAI-RBP1 PAI-1 mRNA-binding protein 1197 C4729 N70455 FBXO31 F-box protein 31 1198 C6623 AA102332 MLSTD1 Male sterility domain containing 1 1199 C8039 Z22970 CD163 CD163 antigen 1200 C8442 AB011151 ZCCHC14 Zinc finger, CCHC domain containing 14 1201 C8606 BC063430 CPXM Carboxypeptidase X (M14 family) 1202 C9730 BQ448187 Transcribed locus 1203 C0791 BC051340 CD164L1 CD164 sialomucin-like 1 1204 C0830 AA012832 IRS1 Insulin receptor substrate 1 1205 C3778 NM_003617 RGS5 Regulator of G-protein signalling 5 1206 C4735 AL136805 ZNF537 Zinc finger protein 537 1207 C4765 CR626993 MRNA; cDNA DKFZp686N07104 (from clone DKFZp686N07104) 1208 C4743 AL137554 CDADC1 Cytidine and dCMP deaminase domain containing 1 1209 C6116 W67536 FLJ31204 Hypothetical protein FLJ31204 1210 C7126 NM_020871 LRCH2 Leucine-rich repeats and calponin homology (CH) domain containing 2 1211 C7172 AF377960 CTTNBP2 Cortactin binding protein 2 1212 C8260 BM981111 MEF2D MADS box transcription enhancer factor 2, polypeptide D (myocyte enhancer factor 2D) 1213 C2020 CA420307 SF3B1 Splicing factor 3b, subunit 1, 155 kDa 1214 C2324 BQ182775 ECRG4 Esophageal cancer related gene 4 protein 1215 C3763 AF480883 PPAP2B Phosphatidic acid phosphatase type 2B 1216 C4549 N64370 TMOD2 Tropomodulin 2 (neuronal) 1217 C6059 AK096344 FLJ35220 Hypothetical protein FLJ35220 1218 C6234 AI247176 TARSH Target of Nesh-SH3 1219 C7503 N90724 IGSF4 Immunoglobulin superfamily, member 4 1220 C7652 AA142913 ARGBP2 Arg/Abl-interacting protein ArgBP2 1221 C7256 NM_021963 NAP1L2 Nucleosome assembly protein 1-like 2 1222 C8088 D87465 SPOCK2 Sparc/osteonectin, cwcv and kazal- like domains proteoglycan (testican) 2 1223 C8286 AY369207 RBPMS2 RNA-binding protein with multiple splicing 2 1224 C8659 BF673156 MYL1 Myosin, light polypeptide 1, alkali; skeletal, fast 1225 D0785 AA936292 Transcribed locus 1226 C0678 AI796508 Transcribed locus 1227 C0274 NM_005157 ABL1 V-abl Abelson murine leukemia viral oncogene homolog 1 1228 C4258 AK092045 C3orf6 Chromosome 3 open reading frame 6 1229 C6974 AK124567 HIBCH 3-hydroxyisobutyryl-Coenzyme A hydrolase 1230 C7876 AW023627 Transcribed locus 1231 C8066 NM_014279 OLFM1 Olfactomedin 1 1232 C9569 H18926 Full-length cDNA clone CS0DK010YA20 of HeLa cells Cot 25-normalized of Homo sapiens (human) 1233 D1322 BX647857 ASB5 Ankyrin repeat and SOCS box- containing 5 1234 C0219 AJ303079 PALM2-AKAP2 PALM2-AKAP2 protein 1235 C1520 BC014640 COL14A1 Collagen, type XIV, alpha 1 (undulin) 1236 C2029 H14510 LOC286191 Hypothetical protein LOC286191 1237 C7654 BC060868 BMPER BMP-binding endothelial regulator precursor protein 1238 C8228 AK124641 CXCL12 Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 1239 C8438 NM_002827 PTPN1 Protein tyrosine phosphatase, non- receptor type 1 1240 C8274 AK056736 MBTPS2 Membrane-bound transcription factor protease, site 2 1241 D0735 AA740582 Transcribed locus 1242 D1185 AA451886 CYP1B1 Cytochrome P450, family 1, subfamily B, polypeptide 1 1243 D1161 BX537988 ST7L Suppression of tumorigenicity 7 like 1244 C0787 AL832207 PLEKHH2 Pleckstrin homology domain containing, family H (with MyTH4 domain) member 2 1245 C3772 U70063 ASAH1 N-acylsphingosine amidohydrolase (acid ceramidase) 1 1246 C3780 AK055619 GNAQ Guanine nucleotide binding protein (G protein), q polypeptide 1247 C4232 AI668702 Transcribed locus 1248 C4884 AA036952 Gup1 GRINL1A complex upstream protein 1249 C6084 W60379 Transcribed locus 1250 C7138 BM678096 TNA Tetranectin (plasminogen binding protein) 1251 C7919 X79981 CDH5 Cadherin 5, type 2, VE-cadherin (vascular epithelium) 1252 C8117 BE905862 SPAG9 Sperm associated antigen 9 1253 C9367 AL558594 PRKAG2 Protein kinase, AMP-activated, gamma 2 non-catalytic subunit 1254 C1466 H03229 GAB1 GRB2-associated binding protein 1 1255 C4732 CR595618 BRMS1L Breast cancer metastasis-suppressor 1-like 1256 C5118 AL137572 C1orf24 Chromosome 1 open reading frame 24 1257 C6476 AA001390 KIAA1463 KIAA1463 protein 1258 C8044 NM_004430 EGR3 Early growth response 3 1259 C7793 BX648935 TBL1XR1 Transducin (beta)-like 1X-linked receptor 1 1260 C8090 NM_032088 PCDHGC3 Protocadherin gamma subfamily C, 3 1261 C4066 AF303058 NP25 Neuronal protein 1262 C3648 AK023450 ANTXR2 Anthrax toxin receptor 2 1263 C8253 BC017021 MEOX2 Mesenchyme homeo box 2 (growth arrest-specific homeo box) 1264 C8456 AB032954 KIAA1128 KIAA1128 1265 C7882 NM_013261 PPARGC1A Peroxisome proliferative activated receptor, gamma, coactivator 1, alpha 1266 C8636 CR607734 DKK3 Dickkopf homolog 3 (Xenopus laevis) 1267 C0671 NM_007197 FZD10 Frizzled homolog 10 (Drosophila) 1268 C1984 W92438 Hypothetical gene supported by BX647608 1269 C2154 AF007144 DIO2 Deiodinase, iodothyronine, type II 1270 C4105 XM_376503 ENPP4 Ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) 1271 C4755 Z30137 LDB3 LIM domain binding 3 1272 C4873 BX537721 CMYA5 Cardiomyopathy associated 5 1273 C7657 AI219521 AP1G1 Adaptor-related protein complex 1, gamma 1 subunit 1274 C8046 NM_002864 PZP Pregnancy-zone protein 1275 C9503 AA621124 LOC338773 Hypothetical protein LOC338773 1276 C3775 BQ003524 KCTD12 Potassium channel tetramerisation domain containing 12 1277 C4082 BC041798 POLK Polymerase (DNA directed) kappa 1278 C4778 N67331 SEC63 SEC63-like (S. cerevisiae) 1279 C6278 BC039245 SART2 Squamous cell carcinoma antigen recognized by T cells 2 1280 C6460 W96022 Transcribed locus 1281 C8119 NM_002775 PRSS11 Protease, serine, 11 (IGF binding) 1282 C9118 BX114286 CD99 CD99 antigen 1283 C8687 NM_006166 NFYB Nuclear transcription factor Y, beta 1284 C9565 AK129819 LHFP Lipoma HMGIC fusion partner 1285 C0284 AK123940 MGC34648 Hypothetical protein MGC34648 1286 C0728 AK095472 DKFZp762C1112 Hypothetical protein DKFZp762C1112 1287 B9925 BC039242 TM4SF10 Transmembrane 4 superfamily member 10 1288 C0579 BX648468 DKFZP564J0863 DKFZP564J0863 protein 1289 C1100 BG257592 FAIM Fas apoptotic inhibitory molecule 1290 C7731 AF245505 DKFZp564I1922 Adlican 1291 C7977 AL833463 LOC283658 Hypothetical protein LOC283658 1292 C7992 AL833291 CMYA3 Cardiomyopathy associated 3 1293 C9580 AK057189 NOX4 NADPH oxidase 4 1294 C9221 CR613361 RNF24 Ring finger protein 24 1295 B9888 AB023155 NAV3 Neuron navigator 3 1296 C4526 N63752 MPHOSPH1 M-phase phosphoprotein 1 1297 C4850 BC040502 BVES Blood vessel epicardial substance 1298 C8203 NM_012180 FBXO8 F-box protein 8 1299 D1419 NM_018328 MBD5 Methyl-CpG binding domain protein 5 1300 C0544 BX640884 C14orf24 Chromosome 14 open reading frame 24 1301 C1403 N49231 KIAA1345 KIAA1345 protein 1302 C6728 AK000337 GFOD1 Glucose-fructose oxidoreductase domain containing 1 1303 C7585 AK095271 LOC128977 Hypothetical protein LOC128977 1304 C7687 CB119523 IL6ST Interleukin 6 signal transducer (gp130, oncostatin M receptor) 1305 C9008 D82786 TA-PP2C T-cell activation protein phosphatase 2C 1306 C0893 BC052210 GARP Glycoprotein A repetitions predominant 1307 C2259 CA436350 Transcribed locus 1308 C3895 BC035090 KPNA3 Karyopherin alpha 3 (importin alpha 4) 1309 C4350 BX089823 FRMD3 FERM domain containing 3 1310 C7057 H22566 DACH1 Dachshund homolog 1 (Drosophila) 1311 C7574 AB028993 NLGN1 Neuroligin 1 1312 C0371 CA431042 Transcribed locus 1313 C0844 BC009951 COLEC11 Collectin sub-family member 11 1314 C1091 AI263903 SIAT10 Sialyltransferase 10 (alpha-2,3- sialyltransferase VI) 1315 C0922 AF378757 PLXDC2 Plexin domain containing 2 1316 C4175 BM683457 EPHA7 EphA7 1317 C4681 XM_071793 C14orf28 Chromosome 14 open reading frame 28 1318 C6523 NM_198968 DZIP1 DAZ interacting protein 1 1319 C6706 BC033034 DIXDC1 DIX domain containing 1 1320 C6572 NM_005197 CHES1 Checkpoint suppressor 1 1321 C8146 BF697545 MGP Matrix Gla protein 1322 C7994 NM_016150 ASB2 Ankyrin repeat and SOCS box- containing 2 1323 C8744 NM_152309 PIK3AP1 Phosphoinositide-3-kinase adaptor protein 1 1324 D0018 BX091065 Transcribed locus 1325 D1273 AJ001015 RAMP2 Receptor (calcitonin) activity modifying protein 2 1326 B9880 CR749402 NFASC Neurofascin 1327 C2088 AF161423 COMMD10 COMM domain containing 10 1328 C4351 CN430728 Transcribed locus 1329 C6723 AA028127 CD209 CD209 antigen 1330 C7059 XM_059702 FLJ36748 Hypothetical protein FLJ36748 1331 C7375 CA312122 PSMC2 Proteasome (prosome, macropain) 26S subunit, ATPase, 2 1332 D0007 CR596214 HNRPA0 Heterogeneous nuclear ribonucleoprotein A0 1333 C0318 M16451 CKB Creatine kinase, brain 1334 C0912 BQ071673 RAMP1 Receptor (calcitonin) activity modifying protein 1 1335 C1412 BX648776 LOC253827 Hypothetical protein LOC253827 1336 C1603 BQ446275 HBD Hemoglobin, delta 1337 C4170 AB007884 ARHGEF9 Cdc42 guanine nucleotide exchange factor (GEF) 9 1338 C4318 AI275068 Transcribed locus 1339 C5860 BU683028 CDNA FLJ10151 fis, clone HEMBA1003402 1340 C5950 CF146489 NKX3-1 NK3 transcription factor related, locus 1 (Drosophila) 1341 C6708 BQ003734 Mesenchymal stem cell protein DSC96 1342 C7078 AK130067 ADAMTS15 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 15 1343 C9587 H17302 LRRC3B Leucine rich repeat containing 3B 1344 D1274 BF435815 MRNA; cDNA DKFZp564O0862 (from clone DKFZp564O0862) 1345 B9884 BX641069 FLJ20481 Hypothetical protein FLJ20481 1346 C1622 AK074184 FLJ34922 Hypothetical protein FLJ34922 1347 C1902 CR591938 WDR33 WD repeat domain 33 1348 C4861 BX647931 Similar to ENSANGP00000004103 1349 C6042 H25761 1350 C6718 AK124339 GJA7 Gap junction protein, alpha 7, 45 kDa (connexin 45) 1351 C7105 R50993 1352 C7439 AA102033 BMPR2 Bone morphogenetic protein receptor, type II (serine/threonine kinase) 1353 D1423 AK055040 MRNA; cDNA DKFZp313C0240 (from clone DKFZp313C0240) 1354 C0357 BC035779 SLC9A9 Solute carrier family 9 (sodium/hydrogen exchanger), isoform 9 1355 C1604 AA044381 1356 C1422 AA095034 GK001 GK001 protein 1357 C2068 XM_375527 LOC339290 Hypothetical protein LOC339290 1358 C3978 BC030112 HIPK3 Homeodomain interacting protein kinase 3 1359 C4287 CR621395 BAG2 BCL2-associated athanogene 2 1360 C6143 NM_001496 GFRA3 GDNF family receptor alpha 3 1361 C5014 AI185804 FN1 Fibronectin 1 1362 C6748 AF487514 GEFT RAC/CDC42 exchange factor 1363 C7089 H14263 GAS1 Growth arrest-specific 1 1364 C8384 X98834 SALL2 Sal-like 2 (Drosophila) 1365 C7744 AF196185 PARD3 Par-3 partitioning defective 3 homolog (C. elegans) 1366 D0657 AB058780 ST6GalII Beta-galactoside alpha-2,6- sialyltransferase II 1367 D0995 BC040983 PCDH7 BH-protocadherin (brain-heart) 1368 C0335 CR590615 ACTA2 Actin, alpha 2, smooth muscle, aorta 1369 C2131 BQ014434 PIAS1 Protein inhibitor of activated STAT, 1 1370 C3746 NM_199511 URB Steroid sensitive gene 1 1371 C4184 NM_020482 FHL5 Four and a half LIM domains 5 1372 C4700 AA934589 MGC45780 Hypothetical protein MGC45780 1373 C7603 AA292234 CDNA FLJ14942 fis, A- PLACE1011185 1374 D1435 T15727 DNCI1 Dynein, cytoplasmic, intermediate polypeptide 1 1375 C2085 AA400893 PDE1A Phosphodiesterase 1A, calmodulin- dependent 1376 C4971 BC000234 NNMT Nicotinamide N-methyltransferase 1377 C7050 AA084479 DNAJC9 DnaJ (Hsp40) homolog, subfamily C, member 9 1378 C9868 AL136646 ARHGAP24 Rho GTPase activating protein 24 1379 B9970 AB014540 SWAP70 SWAP-70 protein 1380 C4998 CR591834 DSTN Destrin (actin depolymerizing factor) 1381 C5058 N62595 KBTBD7 Kelch repeat and BTB (POZ) domain containing 7 1382 C6217 NM_001448 GPC4 Glypican 4 1383 C8023 M81141 HLA-DQB1 Major histocompatibility complex, class II, DQ beta 1 1384 D0533 AF180681 ARHGEF12 Rho guanine nucleotide exchange factor (GEF) 12 1385 D2960 NM_033281 MRPS36 Mitochondrial ribosomal protein S36 1386 D4169 AK128510 GOLPH3 Golgi phosphoprotein 3 (coat- protein) 1387 E0537 BX647115 DPYSL2 Dihydropyrimidinase-like 2 1388 E0690 AI743134 SERPINE2 Serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 1389 D4142 AK091311 JAZF1 Juxtaposed with another zinc finger gene 1 1390 D4211 BC069830 LETM2 Leucine zipper-EF-hand containing transmembrane protein 2 1391 D4328 AK021601 FLJ11539 Hypothetical protein FLJ11539 1392 E1219 AB011175 TBC1D4 TBC1 domain family, member 4 1393 E0783 NM_139033 MAPK7 Mitogen-activated protein kinase 7 1394 D3166 AK097340 RPESP RPE-spondin 1395 D3356 NM_014829 DDX46 DEAD (Asp-Glu-Ala-Asp) box polypeptide 46 1396 D7420 AK124757 SHPRH SNF2 histone linker PHD RING helicase 1397 D6809 AA927082 Transcribed locus 1398 D4165 AK123831 LOC149832 Hypothetical protein LOC149832 1399 D4020 AA858162 C18orf4 Chromosome 18 open reading frame 4 1400 D8933 BX538309 MAMDC2 MAM domain containing 2 1401 E0644 NM_000610 CD44 CD44 antigen (homing function and Indian blood group system) 1402 D4215 AB096175 SP5 Sp5 transcription factor 1403 D5074 AA044778 CDNA FLJ38215 fis, clone FCBBF2000291 1404 D7205 AI040887 ARHGEF7 Rho guanine nucleotide exchange factor (GEF) 7 1405 D8143 AK075059 GLIS3 GLIS family zinc finger 3 1406 E1492 AY326464 TXNDC5 Thioredoxin domain containing 5 1407 D4128 NM_173060 CAST Calpastatin 1408 D4739 BC022957 C9orf102 Chromosome 9 open reading frame 102 1409 E0358 AK021543 DNM3 Dynamin 3 1410 E1300 BC040974 PDE2A Phosphodiesterase 2A, cGMP- stimulated 1411 D9372 AI034385 SORBS1 Sorbin and SH3 domain containing 1 1412 E0240 NM_020433 JPH2 Junctophilin 2 1413 E0721 AW024176 FBLN1 Fibulin 1 1414 D7305 BX092512 SCNN1A Sodium channel, nonvoltage-gated 1 alpha 1415 E0139 AL390147 FAM20C Family with sequence similarity 20, member C 1416 D1798 AK074734 FCGRT Fc fragment of IgG, receptor, transporter, alpha 1417 D3702 AL096748 ARMC8 Armadillo repeat containing 8 1418 D4501 CA447839 FAM49A Family with sequence similarity 49, member A 1419 D5553 AA031882 Transcribed locus 1420 D9082 NM_052954 CYYR1 Cysteine and tyrosine-rich 1 1421 E0475 CR627373 EIF4EBP2 Eukaryotic translation initiation factor 4E binding protein 2 1422 D6213 AK123531 CDNA FLJ41537 fis, clone BRTHA2017985 1423 D9915 BM463727 MEIS4 Meis1, myeloid ecotropic viral integration site 1 homolog 4 (mouse) 1424 E0985 NM_001343 DAB2 Disabled homolog 2, mitogen- responsive phosphoprotein (Drosophila) 1425 D1810 NM_002373 MAP1A Microtubule-associated protein 1A 1426 D4231 C05897 ARL5 ADP-ribosylation factor-like 5 1427 D8491 NM_001122 ADFP Adipose differentiation-related protein 1428 D9934 CA450275 FREQ Frequenin homolog (Drosophila) 1429 E0476 AF000984 DDX3Y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked 1430 E0733 NM_004684 SPARCL1 SPARC-like 1 (mast9, hevin) 1431 E0861 BX648282 ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 1432 D1811 AK128814 CDNA FLJ25106 fis, clone CBR01467 1433 D4059 BF512606 Transcribed locus 1434 D5243 AK074301 FAM8A1 Family with sequence similarity 8, member A1 1435 D6180 AK096674 C14orf32 Chromosome 14 open reading frame 32 1436 D7516 AI074524 DKFZp434H2111 Hypothetical protein DKFZp434H2111 1437 E0726 AB023199 WDR37 WD repeat domain 37 1438 E1622 NM_001753 CAV1 Caveolin 1, caveolae protein, 22 kDa 1439 E1419 AL833496 TAF10 TAF10 RNA polymerase II, TATA box binding protein (TBP)- associated factor, 30 kDa 1440 D7796 CR613362 ALDH6A1 Aldehyde dehydrogenase 6 family, member A1 1441 D8862 NM_032105 PPP1R12B Protein phosphatase 1, regulatory (inhibitor) subunit 12B 1442 E0880 AK000617 LOC92912 Hypothetical protein LOC92912 1443 D5244 BF510155 GPR155 G protein-coupled receptor 155 1444 D7997 AW152624 AKAP13 A kinase (PRKA) anchor protein 13 1445 D8515 CR591759 LUM Lumican 1446 E0237 AI093257 Transcribed locus 1447 E0764 AF087902 TDE2 Tumor differentially expressed 2 1448 E0896 BC045606 NID Nidogen (enactin) 1449 D1767 BC014357 CCND2 Cyclin D2 1450 D4996 NM_001001927 MTUS1 Mitochondrial tumor suppressor 1 1451 D9075 AL832156 CPEB1 Cytoplasmic polyadenylation element binding protein 1 1452 E0623 AL162079 SLC16A1 Solute carrier family 16 (monocarboxylic acid transporters), member 1 1453 D6606 AI733562 Transcribed locus 1454 E0082 AI082254 Transcribed locus 1455 E1421 BC044777 DJ971N18.2 Hypothetical protein DJ971N18.2 1456 D5395 BX094351 Transcribed locus 1457 F0968 AK025758 NFATC2 Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 1458 F1046 NM_014583 LMCD1 LIM and cysteine-rich domains 1 1459 F1770 AK025713 DHX40 DEAH (Asp-Glu-Ala-His) box polypeptide 40 1460 F3132 AL133095 C14orf103 Chromosome 14 open reading frame 103 1461 F3574 NM_016377 AKAP7 A kinase (PRKA) anchor protein 7 1462 F3080 NM_006633 IQGAP2 IQ motif containing GTPase activating protein 2 1463 F9005 D50406 RECK Reversion-inducing-cysteine-rich protein with kazal motifs 1464 F2203 AK024352 EPHA3 EphA3 1465 A7714 AB002351 DMN Desmuslin 1466 C6534 AI057000 Transcribed locus 1467 F1119 U27460 UGP2 UDP-glucose pyrophosphorylase 2 1468 F1176 AY368150 KIAA1228 KIAA1228 protein 1469 F5819 BQ671518 EEF2K Similar to NAD(P) dependent steroid dehydrogenase-like 1470 F6592 AY358353 STK32B Serine/threonine kinase 32B 1471 A6371 BU681010 Full length insert cDNA clone YT94E02 1472 F0018 NM_000963 PTGS2 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) 1473 C0081 NM_182485 CPEB2 Cytoplasmic polyadenylation element binding protein 2 1474 F3496 AB023148 KIAA0931 KIAA0931 protein 1475 F4227 AK001050 C10orf118 Chromosome 10 open reading frame 118 1476 F8898 BE841307 HRMT1L1 HMT1 hnRNP methyltransferase- like 1 (S. cerevisiae) 1477 F3184 NM_033380 COL4A5 Collagen, type IV, alpha 5 (Alport syndrome) 1478 A1331N NM_199072 HIC I-mfa domain-containing protein 1479 A2869 AF054839 TSPAN-2 Tetraspan 2 1480 B4350N AF037364 PNMA1 Paraneoplastic antigen MA1 1481 F1120 L13463 RGS2 Regulator of G-protein signalling 2, 24 kDa 1482 F4886 AK026403 TLN2 Talin 2 1483 F6054 AA905353 NCBP1 Nuclear cap binding protein subunit 1, 80 kDa 1484 F6595 AW938336 CDNA FLJ26188 fis, clone ADG04821 1485 F6738 AK022173 LAF4 Lymphoid nuclear protein related to AF4 1486 B2123 NM_005912 MC4R Melanocortin 4 receptor 1487 C8826 AI091545 SYNCRIP Synaptotagmin binding, cytoplasmic RNA interacting protein 1488 F0480 NM_015635 DKFZP434C212 DKFZP434C212 protein 1489 F2307 AF010236 SGCD Sarcoglycan, delta (35 kDa dystrophin-associated glycoprotein) 1490 F2424 AF111783 MYH4 Myosin, heavy polypeptide 4, skeletal muscle 1491 F4281 AF199023 PLSCR4 Phospholipid scramblase 4 1492 F4410 AK026500 HPCAL1 Hippocalcin-like 1 1493 F7115 AF230201 C20orf17 Chromosome 20 open reading frame 17 1494 F0344 AL049957 CD59 CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, EJ16, EJ30, EL32 and G344) 1495 A7732 BC017984 ARG99 ARG99 protein 1496 A3113 M60445 HDC Histidine decarboxylase 1497 B8326 AK125533 BNIP2 BCL2/adenovirus E1B 19 kDa interacting protein 2 1498 B7331 H45412 EHD2 EH-domain containing 2 1499 F0196 AL050224 PTRF Polymerase I and transcript release factor 1500 F0299 NM_145693 LPIN1 Lipin 1 1501 F0211 BC032379 TMEM18 Transmembrane protein 18 1502 F0307 D86425 NID2 Nidogen 2 (osteonidogen) 1503 F3849 AF302502 PELI2 Pellino homolog 2 (Drosophila) 1504 F5852 AL137573 1505 F9522 AB011141 ZFHX1B Zinc finger homeobox 1b 1506 A0029N BC063856 SPRY1 Sprouty homolog 1, antagonist of FGF signaling (Drosophila) 1507 B6193N NM_030806 C1orf21 Chromosome 1 open reading frame 21 1508 C9234 AK093732 CDNA FLJ36413 fis, clone THYMU2010816 1509 F0121 AF089854 TU3A TU3A protein 1510 F0862 AK023375 CDNA FLJ13313 fis, clone OVARC1001489 1511 F1093 AY029191 ASPN Asporin (LRR class 1) 1512 F3501 AK021708 PDZRN3 PDZ domain containing RING finger 3 1513 F8152 AI022632 RAB7 RAB7, member RAS oncogene family 1514 A3817N AB000114 OMD Osteomodulin 1515 B3754 BC011561 HEPH Hephaestin 1516 C6614 AK074076 USP47 Ubiquitin specific protease 47 1517 F0343 AK025548 TLOC1 Translocation protein 1 1518 F0615 NM_007173 PRSS23 Protease, serine, 23 1519 F2076 AL162032 GPR133 G protein-coupled receptor 133 1520 F3313 AK025164 FLJ21511 Hypothetical protein FLJ21511 1521 A6689 BU741863 SPOCK Sparc/osteonectin, cwcv and kazal- like domains proteoglycan (testican) 1522 B6462 NM_032515 BOK BCL2-related ovarian killer 1523 C9237 XM_211958 1524 C0484 NM_005472 KCNE3 Potassium voltage-gated channel, Isk-related family, member 3 1525 F0482 AK000008 BHMT2 Betaine-homocysteine methyltransferase 2 1526 F0528 AK025661 LIMS1 LIM and senescent cell antigen-like domains 1 1527 F0920 AF098269 PCOLCE2 Procollagen C-endopeptidase enhancer 2 1528 F2225 AF188700 AFAP Hypothetical protein LOC254848 1529 F1525 M24736 SELE Selectin E (endothelial adhesion molecule 1) 1530 F2310 AB002367 DCAMKL1 Doublecortin and CaM kinase-like 1 1531 F3502 X05409 ALDH2 Aldehyde dehydrogenase 2 family (mitochondrial) 1532 F5279 L76566 HLA-DRB6 Major histocompatibility complex, class II, DR beta 6 (pseudogene) 1533 F6175 AV700633 FLJ10404 Hypothetical protein FLJ10404 1534 F6365 AL080114 C10orf72 Chromosome 10 open reading frame 72 1535 F3573 NM_172171 CAMK2G Calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma 1536 D8979 AA740585 1537 F1289 CR623543 SC4MOL Sterol-C4-methyl oxidase-like 1538 F1221 AL109700 CDNA FLJ37610 fis, clone BRCOC2011398 1539 F3457 AB020630 PPP1R16B Protein phosphatase 1, regulatory (inhibitor) subunit 16B 1540 F6060 AK023814 FLJ41603 FLJ41603 protein 1541 A1022N M98399 CD36 CD36 antigen (collagen type I receptor, thrombospondin receptor) 1542 B6200N M79123 NAP1L5 Nucleosome assembly protein 1-like 5 1543 F0927 AK021823 TRIM44 Tripartite motif-containing 44 1544 A1403 J05401 CKMT2 Creatine kinase, mitochondrial 2 (sarcomeric) 1545 B4088N NM_000311 PRNP Prion protein (p27-30) (Creutzfeld- Jakob disease, Gerstmann-Strausler- Scheinker syndrome, fatal familial insomnia) 1546 F0304 NM_002510 GPNMB Glycoprotein (transmembrane) nmb 1547 F1405 AF131837 SIAT7E Sialyltransferase 7 ((alpha-N- acetylneuraminyl-2,3-beta- galactosyl-1,3)-N-acetyl galactosaminide alpha-2,6- sialyltransferase) E 1548 F1225 AF118108 XLKD1 Extracellular link domain containing 1 1549 F4131 AF389429 SEMA6D Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D 1550 C8476 R59552 CHRDL1 Chordin-like 1 1551 C1827 BC008703 TCEAL3 Transcription elongation factor A (SII)-like 3 1552 D6878 AI002365 PDGFRB Platelet-derived growth factor receptor, beta polypeptide 1553 D7732 CB242274 Transcribed locus 1554 F2379 AB002365 KIAA0367 KIAA0367 1555 F1446 AJ277587 SPIRE1 Spire homolog 1 (Drosophila) 1556 F3692 NM_004673 ANGPTL1 Angiopoietin-like 1 1557 A1101N NM_022977 ACSL4 Acyl-CoA synthetase long-chain family member 4 1558 B7430N AA522674 LIMS2 LIM and senescent cell antigen-like domains 2 1559 B7571N BU619137 TGFBR3 Transforming growth factor, beta receptor III (betaglycan, 300 kDa) 1560 F1143 AF070543 ODZ2 Odz, odd Oz/ten-m homolog 2 (Drosophila) 1561 F1241 AF114263 HH114 Hypothetical protein HH114 1562 F2686 CR616854 EVI2B Ecotropic viral integration site 2B 1563 F2462 NM_182734 PLCB1 Phospholipase C, beta 1 (phosphoinositide-specific) 1564 F2715 BC035776 CILP Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase 1565 F4824 U82319 YDD19 YDD19 protein 1566 A0203N NM_000043 TNFRSF6 Tumor necrosis factor receptor superfamily, member 6 1567 A5933 XM_059689 Similar to CG4502-PA 1568 C0524 BM724780 Transcribed locus, weakly similar to XP_375099.1 hypothetical protein LOC283585 [Homo sapiens] 1569 C8150 NM_014335 CRI1 CREBBP/EP300 inhibitor 1 1570 C9677 AL832661 LOC143381 Hypothetical protein LOC143381 1571 E2113 BC005248 EIF1AY Eukaryotic translation initiation factor 1A, Y-linked 1572 F0821 AL050030 1573 F2190 AK021985 FBXL7 F-box and leucine-rich repeat protein 7 1574 F1447 NM_014629 ARHGEF10 Rho guanine nucleotide exchange factor (GEF) 10 1575 F2205 AF052181 EPIM Epimorphin 1576 F0092 AK001789 SMUG1 Single-strand selective monofunctional uracil DNA glycosylase 1577 A3258 U19487 PTGER2 Prostaglandin E receptor 2 (subtype EP2), 53 kDa 1578 B4152N W89185 SET7 SET domain-containing protein 7 1579 B8840 BX648004 SPG20 Spastic paraplegia 20, spartin (Troyer syndrome) 1580 F1146 AK025893 RBPMS RNA binding protein with multiple splicing 1581 F0597 AK000146 CGI-30 CGI-30 protein 1582 F2464 AK027243 BBS1 Bardet-Biedl syndrome 1 1583 F2803 AF170562 USP25 Ubiquitin specific protease 25 1584 F4063 AL109779 HDGFRP3 Hepatoma-derived growth factor, related protein 3 1585 F4950 NM_194430 RNASE4 Angiogenin, ribonuclease, RNase A family, 5 1586 F7716 BE178490 Hypothetical gene supported by AK093334; AL833330; BC020871; BC032492 1587 A0095 J03241 TGFB3 Transforming growth factor, beta 3 1588 A0217 M83233 TCF12 Transcription factor 12 (HTF4, helix-loop-helix transcription factors 4) 1589 F0001N NM_153831 PTK2 PTK2 protein tyrosine kinase 2 1590 A0375N BC057815 RRAD Ras-related associated with diabetes 1591 A0911N M63256 CDR2 Cerebellar degeneration-related protein 2, 62 kDa 1592 C1400 BC007632 KIAA0318 RIM binding protein 2 1593 C0661 H18687 CLDN11 Claudin 11 (oligodendrocyte transmembrane protein) 1594 C8718 AA206141 PRICKLE1 Prickle-like 1 (Drosophila) 1595 D6726 AA897762 PPM1A Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform 1596 F3564 CR749667 PDE4B Phosphodiesterase 4B, cAMP- specific (phosphodiesterase E4 dunce homolog, Drosophila) 1597 F4186 AB023168 NLGN4Y Neuroligin 4, Y-linked 1598 F6116 BC030244 TNNC1 Troponin C, slow 1599 F7080 AW973637 GGTA1 Glycoprotein, alpha- galactosyltransferase 1 1600 F7477 AW868740 SYNPO2 Synaptopodin 2 1601 F0520 BC041337 RHOBTB3 Rho-related BTB domain containing 3 1602 B4181 AK021510 KCNMB3 Potassium large conductance calcium-activated channel, subfamily M beta member 3 1603 B5089N AA828067 C1QB Complement component 1, q subcomponent, beta polypeptide 1604 B7158N XM_085175 TTC7B Tetratricopeptide repeat domain 7B 1605 E1632 BU633335 SMAD4 SMAD, mothers against DPP homolog 4 (Drosophila) 1606 F0174 AK024029 MOAP1 Modulator of apoptosis 1 1607 F1147 AK125336 LOC90167 Hypothetical protein LOC90167 1608 F4952 AL080082 MRNA; cDNA DKFZp564G1162 (from clone DKFZp564G1162) 1609 B3745 N92541 Transcribed locus 1610 A0270N AF241831 HABP4 Hyaluronan binding protein 4 1611 B6485 BC009753 ACACB Acetyl-Coenzyme A carboxylase beta 1612 F0416 AF082557 TNKS Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 1613 F0470 AJ250865 TES Testis derived transcript (3 LIM domains) 1614 F0911 L08177 EBI2 Epstein-Barr virus induced gene 2 (lymphocyte-specific G protein- coupled receptor) 1615 F2392 NM_001901 CTGF Connective tissue growth factor 1616 F3618 AK172810 SLC39A14 Solute carrier family 39 (zinc transporter), member 14 1617 F4440 AB032773 TU12B1-TY TU12B1-TY protein 1618 F6326 NM_015458 MTMR9 Myotubularin related protein 9 1619 F7458 AK123706 ADAMTS8 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 8 1620 A1666N NM_000176 NR3C1 Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) 1621 A3471 NM_006281 STK3 Serine/threonine kinase 3 (STE20 homolog, yeast) 1622 B7499 BX641020 ARID5B AT rich interactive domain 5B (MRF1-like) 1623 B8216 CR623023 Full-length cDNA clone CS0DC029YI23 of Neuroblastoma Cot 25-normalized of Homo sapiens (human) 1624 F0286 NM_000132 F8 Coagulation factor VIII, procoagulant component (hemophilia A) 1625 F1338 AF056195 NAG Neuroblastoma-amplified protein 1626 F2115 AK021795 BNC2 Basonuclin 2 1627 F2699 AF022789 USP12 Ubiquitin specific protease 12 1628 F5702 AK024358 MPEG1 Macrophage expressed gene 1 1629 A0279 NM_005257 GATA6 GATA binding protein 6 1630 A3940 AF048722 PITX2 Paired-like homeodomain transcription factor 2 1631 F0004 NM_005252 FOS V-fos FBJ murine osteosarcoma viral oncogene homolog 1632 G2550 NM_000962 PTGS1 Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) 1633 B2787 CR619015 MRGPRF MAS-related GPR, member F 1634 B6424 AL049313 CLIC5 Chloride intracellular channel 5 1635 C0810 AK123757 EBF Early B-cell factor 1636 F2322 AL080213 PDE4DIP Phosphodiesterase 4D interacting protein (myomegalin) 1637 F1600 AB038523 MBIP MAP3K12 binding inhibitory protein 1 1638 F2393 M14091 SERPINA7 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 7 1639 F2908 AK023821 MACF1 Microtubule-actin crosslinking factor 1 1640 A8838 AK075242 MGC45438 Hypothetical protein MGC45438 1641 B4271 AB011121 ALS2CR3 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 3 1642 B7509 CN268436 CDNA clone IMAGE: 5263177, partial cds 1643 B9025 BU537728 HSA9761 Putative dimethyladenosine transferase 1644 F0288 BC080187 LMOD1 Leiomodin 1 (smooth muscle) 1645 F0333 AK026095 SNTB1 Syntrophin, beta 1 (dystrophin- associated protein A1, 59 kDa, basic component 1) 1646 F0728 NM_021614 KCNN2 Potassium intermediate/small conductance calcium-activated channel, subfamily N, member 2 1647 F1259 AK000776 Full-length cDNA clone CS0DD009YB17 of Neuroblastoma Cot 50-normalized of Homo sapiens (human) 1648 F2702 AL049990 MRNA; cDNA DKFZp564G112 (from clone DKFZp564G112) 1649 F4940 BC035161 CRY2 Cryptochrome 2 (photolyase-like) 1650 A4080 AF054992 PKD2 Polycystic kidney disease 2 (autosomal dominant) 1651 A9898 BC010353 PTPLA Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member a 1652 B1461N CR744550 MYO9A Myosin IXA 1653 C0591 AB014523 ULK2 Unc-51-like kinase 2 (C. elegans) 1654 C1450 AB075828 ZNF545 Zinc finger protein 545 1655 F0518 AF035307 PLXNC1 Plexin C1 1656 F2283 AJ276316 ZNF304 Zinc finger protein 304 1657 F2335 AK001832 FLJ10970 Hypothetical protein FLJ10970 1658 F5448 AK023831 FLJ13769 Hypothetical protein FLJ13769 1659 F2161 AF116646 GALNACT-2 Chondroitin sulfate GalNAcT-2 1660 A1157N NM_002667 PLN Phospholamban 1661 B4276 AK056725 CDNA FLJ32163 fis, clone PLACE6000371 1662 B5186N AK056963 Full length insert cDNA clone ZE03F06 1663 C6412 BX090035 Transcribed locus 1664 F0182 BC009203 LOC90355 Hypothetical gene supported by AF038182; BC009203 1665 F1343 BC032404 DKFZp434D0215 SH3 domain protein D19 1666 F4628 AF119893 Identification of C2093, B5860N and C6055 as Up-Regulated Genes in Bladder Cancer Cells

When gene-expression profiles of cancer cells from 33 bladder cancer patients were analyzed using a cDNA microarray representing 27,648 human genes, 394 genes that were commonly up-regulated in bladder cancer cells were identified. Among them, attention was focused on the genes with the in-house codes C2093, which designated M-phase phosphoprotein 1 (MPHOSPH1) (Genebank Accession NM_(—)016195 (SEQ ID NO. 1, encoding SEQ ID NO. 2)), B5860N, designated DEP domain containing 1 (DEPDC1) (SEQ ID NO. 3, encoding SEQ ID NO. 4), and C6055, designated MGC34032 hypothetical protein, (Genebank Accession NM_(—)152697 SEQ ID NO: 133, encoding SEQ ID NO: 134). Expression of the C2093, B5860N and C6055 genes were elevated in 24 of 25, 17 of 20 and 21 of 32 bladder cancer cases which were able to obtain expression data, respectively. To confirm the expression of these up-regulated genes, semi-quantitative RT-PCR analysis was performed to compare the expression level between bladder cancer specimens and normal human tissues including normal bladder cancer cells. Firstly, it was discovered that C2093 showed the elevated expression in 17 of 21 clinical bladder cancer samples, as compared to normal bladder cells and normal human tissues including lung, heart, liver and kidney (FIGS. 1 a and b). In addition, this gene was overexpressed in all of six bladder cancer cell lines as well (FIG. 1 b). Next, it was discovered that B5860N showed the elevated expression in 20 of 21 clinical bladder cancer specimens compared to normal human tissues, especially normal bladder mRNA (FIGS. 1 a and c), and was overexpressed in all of six bladder cancer cell lines we examined (FIG. 1 c).

To further examine the expression pattern of these genes, northern blot analyses were performed with multiple-human tissues and bladder cancer cell lines using cDNA fragments of C2093, B5860N and C6055 as probes (see Material and Method). Expression of C2093 was no or undetectable in normal human tissues except testis (FIG. 2 e; the upper panel), while was surprisingly overexpressed in all of bladder cancer cell lines (FIG. 2 e; the bottom panel). B5860N was also exclusively expressed in testis (FIG. 2 f, the upper panel), while was significantly overexpressed in all of bladder cancer cell lines, compared to in other normal tissues, especially in normal human bladder (FIG. 2 f, the bottom panel). C6055 was also no or undetectable in normal human tissues (FIG. 2 g; the upper panel), while was overexpressed in three of six bladder cancer cell lines (FIG. 2 g; the bottom panel). Thus, attention was focused the bladder cancer specifically expressed transcripts.

Genomic Structure of C2093, B5860N and C6055

To obtain the entire cDNA sequences of C2093, B5860N and C6055, RT-PCR was performed as EST-walking, and 5′RACE and 3′RACE experiments using bladder cancer cell line, SW780, as template (see Materials and Methods) because C2093 initially was not full length on database. C2093 consists of 31 exons, designated M-phase phosphoprotein 1 (MPHOSPH1), located on the chromosome 10q23.31. The full-length mRNA sequences of C2093 contained 6319 nucleotides, encoding 1780 amino acids. The ORF of this transcript starts at within each exon 1.

B5860N, designated DEP domain containing 1 (DEPDC1), located on the chromosome 1p31.2. This gene has also two different transcriptional variants consisting of 12 and 11 exons, corresponding to B5860N V1 (SEQ ID NO. 3, encoding SEQ ID NO. 4) and B5860N V2 (SEQ ID NO. 5, encoding SEQ ID NO. 6), respectively (FIG. 3 b). There were alternative variations in exon 8 of V1, and the other remaining exons were common to both variants. V2 variant has no exon 8 of the V1, generating same stop codon within last exon. The full-length cDNA sequences of B5860NV1 and B5860NV2 variants consist of 5318 and 4466 nucleotides, respectively. The ORF of these variants start at within each exon 1. Eventually, V1 and V2 transcripts encode 811 and 527 amino acids, respectively. To further confirm the expression pattern of each variant in bladder cancer cell lines and normal human tissues including bladder, heart, lung, liver, kidney, brain, pancreas, northern blot analysis was performed. As a result, it was discovered that both variants were highly overexpressed in bladder cancer cells, but no or undetectable expression in normal human tissues (FIG. 2 f, lower panel) except trestis. In particular, V2 transcript was expressed exclusively in testis. Therefore, functional analysis for both variants of B5860N were further performed.

According to the database from NCBI, C6055 consists of 24 exons, designated MGC34032, located on the chromosome 1p31.3. Because C6055 is not included within last exon (exon 24) of MGC34032 on database, we performed RT-PCR as EST-walking, and 5′RACE experiments using bladder cancer cell line, SW780, as a template to obtain the entire cDNA sequence of C6055 (see Materials and Methods). As a result, we found two novel transcripts, C6055V1 (SEQ ID NO: 129, encoding SEQ ID NO: 130) and C6055V2 (SEQ ID NO: 131, encoding SEQ ID NO: 132). Eventually, this gene has four different splicing variants consisting of 24, 25, 22 and 22 exons, corresponding to MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2, respectively (FIG. 3 c). There were alternative splicing in exon 1, 2, 3, 4 and 24 of MGC34032, and the other remaining exons were common among four transcripts. C6055V1 and C6055V2 transcripts have no exon 1, 2 and 3 of MGC34032, generating same stop codon within last exon. In particular, the ORF of C6055V1 and C6055V2 transcripts start at within each exon 4, indicating C6055V1 and C6055V2 transcripts have same ORF. The full-length cDNA sequences of MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2 transcripts consist of 2302, 3947, 3851, and 3819 nucleotides, respectively. Eventually, MGC34032, Genbank Accession No. AK128063, C6055V1 and C6055V2 transcripts encode 719, 587, 675 and 675 amino acids, respectively. To further confirm the expression pattern of each variant in bladder cancer cell lines and normal human tissues including bladder, heart, lung, liver, kidney, brain, testis, pancreas, we performed northern blot analysis using a cDNA fragment C6055 for microarray as a probe. As a result, approximately 3.9 kb transcripts were highly overexpressed in some bladder cancer cells (HT-1376, SW780 and RT4), but no or undetectable expression in normal human tissues (FIG. 2 g upper panel). In addition, 7.5 kb transcript was specifically expressed only in HT1376 cells, but we have not yet identified the entire mRNA sequence of this transcript. Furthermore, when we performed northern blot analysis using the common region among these transcripts as a probe, we detected 2.3 kb transcript exclusively in normal testis, corresponding to MGC34032 (FIG. 2 g middle and lower panel). Therefore, we further perform functional analysis for C6055V1 gene product.

Subcellular Localization of C2093, B5860N and C6055

To further examine the characterization of C2093, B5860N and C6055, the sub-cellular localization of these gene products was examined in COS7 cells. Firstly, when plasmids expressing the C2093 protein (pCAGGS-C2093-HA) were transiently transfected into COS7 cells, the 210 KDa-C2093 protein was observed as an expected size by Western blot analysis (FIG. 4 a). Immunocytochemical staining reveals exogenous C2093 mainly localized to the nucleus apparatus in COS7 cells, but in some cells was observed to be accumulated around chromosome (FIG. 4 b). Therefore, the cells were synchronized using aphidicolin and examine C2093 protein localization during cell-cycle progression. Notably the protein was located in nuclei at G1/G0 and S phases, and, especially, accumulated around chromosome during G2/M phase (FIG. 4 c).

Next, when plasmids expressing B5860NV1 or V2 proteins (pCAGGS-B5860NV1-HA or pCAGGS-B5860NV2-HA) were transiently transfected into COS7, respectively, exogenous B5860NV1 and V2 proteins were observed as each expected size by Western blot analysis at 24 and 48 hours after transfection (FIG. 4 d, V1; left panel, V2 right panel). Moreover, immunocytochemical staining reveals that B5860NV1 localized to the cytoplasm (FIG. 4 e, upper panel), but some cells was also observed to be nuclei apparatus (FIG. 4 e, bottom panel), ant that B5860NV2 mainly localized to the cytoplasm (FIG. 4 f, upper panel), and some cells was also observed under cytoplasmic membrane (FIG. 4 f, bottom panel). Therefore, the cells were synchronized using aphidicolin and examined B5860NV1 and V2 proteins localization during cell-cycle progression. B5860NV1 protein was located in nuclei at G1/G0 and S phases, but localized under cytoplasmic membrane during G2/M phases (FIG. 4 g), but B5860NV2 protein was located under cytoplasmic membrane during G2/M phase (FIG. 4 h). Furthermore, when both plasmids expressing B5860NV1 and V2 proteins were transiently co-transfected into COS7, it was observed that B5860NV1 protein located in nuclei and cytoplasm apparatus, and B5860NV2 located nuclei and translocated to under cytoplasmic membrane during G2/M phase (FIG. 4 i).

To further determine the subcellular localization of endogenous C2093 localization during cell cycle progression by immunocytochemical analysis using affinity-purified anti-C2093 antibodies. Endogenous C2093 protein was localized in the nucleus during interphase, but in the cytoplasm during prophase, metaphase and early anaphase, especially located in the midbody in late anaphase, and then near the contractile ring in telophase (FIG. 4 j). Therefore, C2093 may play an important role in the cytokinesis.

Next, we examined endogenous B5860N in bladder cancer cells during cell cycle progression as well as C2093, we performed immunocytochemical analysis using affinity-purified B5860N polyclonal antibodies. Endogenous B5860N protein was localized mainly in the nucleus during interphase, but in the cytoplasm during M-phase (FIG. 4 k).

The SMART and SOSUI computer predictions revealed that the predicted C6055 protein contained 8th, 9th or 10th transmembrane domains. To confirm this prediction, we examined the sub-cellular localization of this gene product in COS7 cells at 36 and 60 hours after transfection. Firstly, when we transiently transfected plasmids expressing C6055 protein (pCAGGS-C6055-HA) into COS7 cells, we performed Western blot analysis using an anti-HA tag antibody. The results showed a 67 KDa-band corresponding to the predicted size of the C6055 protein as well as an additional 75 KDa band (FIG. 41). To verify whether the 75 KDa band represented a form of C6055 modified by phosphorylation or glycosylation, we treated the cellular extracts with X-phosphatase, O-glycosidase or N-glycosydase and O-glycosidase before immunoblotting. Although the 75-kDa band did not disappear after phosphatase and O-glycosidase, it disappeared after O-glycosidase treatment, suggesting that C6055 protein was O-glycosylated only in living cells (FIG. 4 m). To investigate the subcellular localization of C6055 protein, we performed fluorescent immunohistochemical staining in C6055-transfected COS7 cells. The results revealed exogenous C6055 mainly localized to cytoplasmic membrane in COS7 cells at 60 hours although we observed C6055 localization in cytoplasm at 36 hours (FIG. 4 n).

Growth-Inhibitory Effects of Small-Interfering RNA (siRNA) Designed to Reduce Expression of C2093, B5860N and C6055

To assess the growth-promoting role of C2093, B5860N and C6055, the expression of endogenous C2093, B5860N and C6055 was knocked down in bladder cancer lines, J82, UMUC3 and SW780 that have shown the overexpression of C2093, B5860N and C6055, by means of the mammalian vector-based RNA interference (RNAi) technique (see Materials and Methods). Expression levels of C2093, B5860N and C6055 were examined by semi-quantitative RT-PCR experiments. As shown in FIGS. 5 to 7 C2093, B5860N and C6055-specific siRNAs significantly suppressed expression of each gene, compared with control siRNA constructs (psiU6BX-EGFP and SCR) (FIG. 5 a, 5 d, 6 a, 7 a). To confirm the cell growth inhibition with C2093, B5860N and C6055-specific siRNAs, we performed colony-formation and MTT assays were performed, respectively. Introduction of C2093-si3 constructs suppressed growth of J82 and UMUC3 cells (FIG. 5 b, c, e, f), B5860N-si3 constructs suppressed growth of J82 cells (FIGS. 6 b, c) and C6055-si-08 constructs suppressed growth of SW780 cells (FIG. 7 b,c), consisting with the result of above reduced expression. Each result was verified by three independent experiments. These findings suggest that C2093, B5860N and C6055 have a significant function in the cell growth of the bladder cancer.

In particular, to further elucidate the role of C2093 in cytokinesis, we transfected C2093-siRNA into bladder cancer cell line UMUC3 cells and then observed cell morphology by microscopy on 7 days after transfection. We confirmed expression of C2093 protein was knockdowned by C2093-siRNA (FIG. 8 b), and observed multi-nucleated cells in siRNA-transfected cells (FIG. 8 a, c), indicating si-C2093 knockdown cells failed in cytokinesis.

Expression of C2093 and B6850N Proteins in Clinical Samples.

We performed immunohistochemical analysis of C2093 or B5860N in surgically resected invasive bladder cancer tissue and normal bladder tissue sections and various normal tissues (kidney, heart, lung and liver), respectively. Strong staining against both proteins were observed only in bladder cancer tissues (FIG. 9 a, b), and undetectable staining of C2093 was observed normal bladder tissue (FIG. 9 a).

Discussion:

In this report, through the precise expression profiles of bladder cancer by means of genome wide cDNA microarray, novel genes, C2093, B5860N and C6055 that were significantly overexpressed in bladder cancer cells, as compared to normal human tissues, were isolated.

The B5860N protein was observed to localize in cytoplasm as intermediate filaments by immunochemical staining, suggesting that B5860N may play a key role of interaction of cell to cell.

Furthermore, it was demonstrated that treatment of bladder cancer cells with siRNA effectively inhibited expression of all three target genes, C2093, B5860N and C6055 and significantly suppressed cell/tumor growth of bladder cancer. These findings suggest that C2093, B5860N and C6055 might play key roles in tumor cell growth proliferation, and may be promising targets for development of anti-cancer drugs.

INDUSTRIAL APPLICABILITY

The gene-expression analysis of bladder cancer described herein, obtained through a combination of laser-capture dissection and genome-wide cDNA microarray, has identified specific genes as targets for cancer prevention and therapy. Based on the expression of a subset of these differentially expressed genes, the present invention provides molecular diagnostic markers for identifying and detecting bladder cancer.

The methods described herein are also useful in the identification of additional molecular targets for prevention, diagnosis and treatment of bladder cancer. The data reported herein add to a comprehensive understanding of bladder cancer, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of bladder tumorigenesis, and provide indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of bladder cancer.

The expression of human genes C2093, B5860Ns and C6055s are markedly elevated in bladder cancer as compared to non-cancerous bladder tissue. Accordingly, these genes are useful as a diagnostic marker of bladder cancer and the proteins encoded thereby are useful in diagnostic assays of bladder cancer.

The present inventors have also shown that the expression of the C2093, B5860Ns or C6055s proteins promote cell growth whereas cell growth is suppressed by small interfering RNAs corresponding to the C2093, B5860Ns or C6055s genes. These findings show that C2093, B5860Ns and C6055s proteins stimulates oncogenic activity. Thus, each of these oncoproteins is a useful target for the development of anti-cancer pharmaceuticals. For example, agents that block the expression of C2093, B5860Ns or C6055s, or prevent its activity find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of bladder cancers. Examples of such agents include antisense oligonucleotides, small interfering RNAs, and ribozymes against the C2093, B5860Ns or C6055s gene, and antibodies that recognize C2093, B5860Ns or C6055s.

All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Furthermore, while the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents. 

The invention claimed is:
 1. A method of screening for a compound for treating or preventing bladder cancer, said method comprising the steps of: (a) contacting a test compound with a polypeptide selected from the group consisting of: (1) a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6; (2) a polypeptide that comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6 or a sequence having at least about 80% homology to said sequence; and (3) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 5, wherein the polypeptide has a biological activity equivalent to a polypeptide consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6; (b) detecting the biological activity of the polypeptide of step (a); and (c) selecting a compound that suppresses the biological activity of the polypeptide in comparison with the biological activity detected in the absence of the test compound; wherein the biological activity is cell-proliferating activity in a bladder cancer cell. 